Histone deacetylase inhibitors and process for producing the same

ABSTRACT

Compounds represented by formula (1) have strong inhibitory activity that is selective towards HDAC1 and HDAC4. Therefore, the compounds of the present invention are useful as pharmaceutical agents for treating or preventing diseases caused by HDAC1 and HDAC4.

TECHNICAL FIELD

The present invention relates to histone deacetylase (HDAC) inhibitorsand methods for producing the same.

BACKGROUND ART

Eukaryotic chromatin structure and gene expression are regulated byhistone acetylation by histone acetyltransferase (HAT), anddeacetylation by histone deacetylase (HDAC). HDAC inhibitors are alreadyknown to induce cancer cell differentiation and apoptosis, and areexpected to be useful as antitumor agents (Marks, P. A., Richon, V. M.,and Rifkind, R. A. (2000). Histone deacetylase inhibitors: Inducers ofdifferentiation or apoptosis of transformed cells. J. Natl. Cancer Inst.92, 1210-1216; Yoshida, M., Horinouchi, S., and Beppu, T. (1995).Trichostatin A and trapoxin: novel chemical probes for the role ofhistone acetylation in chromatin structure and function. Bioessays 17,423-430; Bernhard, D., Loffler, M., Hartmann, B. L., Yoshida, M.,Kofler, R., and Csordas, A. (1999). Interaction between dexamethasoneand butyrate in apoptosis induction: non-additive in thymocytes andsynergistic in a T cell-derived leukemia cell line. Cell Death Diff. 6,609-607).

In fact, clinical studies have begun in the United States for some HDACinhibitors (Nakajima, H., Kim, Y. B., Terano, H., Yoshida, M., andHorinouchi, S. (1998). FR901228, a potent antitumor antibiotic, is anovel histone deacetylase inhibitor. Exp. Cell Res. 241, 126-133; Saito,A., Yamashita, T., Mariko, Y., Nosaka, Y., Tsuchiya, K., Ando, T.,Suzuki, T., Tsuruo, T., and Nakanishi, O. (1999). A synthetic inhibitorof histone deacetylase, MS-27-275, with marked in vivo antitumoractivity against human tumors. Proc. Natl. Acad. Sci. USA 96, 4592-4597)that are effective as antitumor agents in animal experiments.

Tricostatin A (TSA) is well known as a specific HDAC inhibitor (Yoshida,M., Kijima, M., Akita, M., and Beppu, T. (1990). Potent and specificinhibition of mammalian histone deacetylase both in vivo and in vitro bytrichostatin A. J. Biol. Chem. 265, 17174-17179). Actually, TSA has beenknown to induce differentiation to leukemia cells, neuronal cells,breast cancer cells, and the like (Yoshida, M., Nomura, S., and Beppu,T. Effects of trichostatins on differentiation of murine erythroleukemiacells. Cancer Res. 47: 3688-3691, 1987; Hoshikawa, Y., Kijima, M.,Yoshida, M., and Beppu, T. Expression of differentiation-related markersin teratocarcinoma cells via histone hyperacetylation by trichostatin A.Agric. Biol. Chem. 55: 1491-1495, 1991; Minucci, S., Horn, V.,Bhattacharyya, N., Russanova, V., Ogryzko, V. V., Gabriele, L., Howard,B. H., and Ozato, K. A histone deacetylase inhibitor potentiatesretinoid receptor action in embryonal carcinoma cells. Proc. Natl. Acad.Sci. USA 94: 11295-11300, 1997; Inokoshi, J., Katagiri, M., Arima, S.,Tanaka, H., Hayashi, M., Kim, Y. B., Furumai, R., Yoshida, M.,Horinouchi, S., and Omura, S. (1999). Neuronal differentiation of Neuro2a cells by inhibitors of cell progression, trichostatin A andbutyrolactone I. Biochem. Biophys. Res. Commun. 256, 372-376; Wang, J.,Saunthararajah, Y., Redner, R. L., and Liu, J. M. Inhibitors of histonedeacetylase relieve ETO-mediated repression and induce differentiationof AML1-ETO leukemia cells. Cancer Res. 59: 2766-2769, 1999; Munster, P.N., Troso-Sandoval, T., Rosen, N., Rifkind, R., Marks, P. A., andRichon, V. M. The histone deacetylase inhibitor suberoylanilidehydroxamic acid induces differentiation of human breast cancer cells.Cancer Res. 61: 8492-8497, 2001; Ferrara, F. F., Fazi, F., Bianchini,A., Padula, F., Gelmetti, V., Minucci, S., Mancini, M., Pelicci, P. G.,Lo Coco, F., and Nervi, C. Histone deacetylase-targeted treatmentrestores retinoic acid signaling and differentiation in acute myeloidleukemia. Cancer Res. 61: 2-7, 2001; Gottlicher, M., Minucci, S., Zhu,P., Kramer, O. H., Schimpf, A., Giavara, S., Sleeman, J. P., Lo Coco,F., Nervi, C., Pelicci, P. G., and Heinzel, T. Valproic acid defines anovel class of HDAC inhibitors inducing differentiation of transformedcells. EMBO J. 20: 6969-6978, 2001). Furthermore, the TSA activities ofdifferentiation induction and apoptosis induction are known tosynergistically increase when used in combination with drugs whichactivate gene expression by mechanisms different to HDAC inhibitors. Forexample, cancer cell differentiation is promoted by using HDACinhibitors in combination with retinoic acids, which activate retinoicacid receptors that serve as nuclear receptors, inducing gene expressionrelevant to differentiation (Minucci, S., Horn, V., Bhattacharyya, N.,Russanova, V., Ogryzko, V. V., Gabriele, L., Howard, B. H., and Ozato,K. A histone deacetylase inhibitor potentiates retinoid receptor actionin embryonal carcinoma cells. Proc. Natl. Acad. Sci. USA 94:11295-11300, 1997; Ferrara, F. F., Fazi, F., Bianchini, A., Padula, F.,Gelmetti, V., Minucci, S., Mancini, M., Pelicci, P. G., Lo Coco, F., andNervi, C. Histone deacetylase-targeted treatment restores retinoic acidsignaling and differentiation in acute myeloid leukemia. Cancer Res. 61:2-7, 2001; Coffey, D. C., Kutko, M. C., Glick, R. D., Butler, L. M.,Heller, G., Rifkind, R. A., Marks, P. A., Richon, V. M., and La Quaglia,M. P. The histone deacetylase inhibitor, CBHA, inhibits growth of humanneuroblastoma xenografts in vivo, alone and synergistically withall-trans retinoic acid. Cancer Res. 61: 3591-3594, 2001; Petti, M. C.,Fazi, F., Gentile, M., Diverio, D., De Fabritiis, P., De Propris, M. S.,Fiorini, R., Spiriti, M. A., Padula, F., Pelicci, P. G., Nervi, C., andLo Coco, F. Complete remission through blast cell differentiation inPLZF/RARalpha-positive acute promyelocytic leukemia: in vitro and invivo studies. Blood 100: 1065-1067, 2002). 5-azadeoxycytidine inhibitsDNA methylation to reduce expression of tumor suppressor genes in manycancer cells. TSA used in combination with 5-azadeoxycytidine promotescancer cell apoptosis and restoration of tumor-suppressing geneexpression (Nan, X., Ng, H. H., Johnson, C. A., Laherty, C. D., Turner,B. M., Eisenman, R. N., and Bird, A. Transcriptional repression bydeacetylase complex. Nature 393: 386-389, 1998; Cameron, E. E., Bachman,K. E., Myohanen, S., Herman, J. G., and Baylin, S. B. Synergy ofdemethylation and histone deacetylase inhibition in the re-expression ofgenes silenced in cancer. Nature Genet. 21: 103-107, 1999; Li, Q. L.,Ito, K., Sakakura, C., Fukamachi, H., Inoue, K., Chi, X. Z., Lee, K. Y.,Nomura, S., Lee, C. W., Han, S. B., Kim, H. M., Kim, W. J., Yamamoto,H., Yamashita, N., Yano, T., Ikeda, T., Itohara, S., Inazawa, J., Abe,T., Hagiwara, A., Yamagishi, H., Ooe, A., Kaneda, A., Sugimura, T.,Ushijima, T., Bae, S. C., and Ito, Y. Causal relationship between theloss of RUNX3 expression and gastric cancer. Cell 109: 113-124, 2002;Boivin, A. J., Momparler, L. F., Hurtubise, A., and Momparler, R. L.Antineoplastic action of 5-aza-2′-deoxycytidine and phenylbutyrate onhuman lung carcinoma cells. Anticancer Drugs 13: 869-874, 2002; Primeau,M., Gagnon, J., and Momparler, R. L. Synergistic antineoplastic actionof DNA methylation inhibitor 5-AZA-2′-deoxycytidine and histonedeacetylase inhibitor depsipeptide on human breast carcinoma cells. IntJ Cancer 103: 177-184, 2003).

HDAC inhibitors are expected to be not only antitumor agents but alsocancer preventives. TSA, SAHA, and the like significantly suppressed theoccurrence of breast cancer induced in animal models. Also,investigations carried out using valproic acids indicated that HDACinhibitors suppress metastasis (Gottlicher, M., Minucci, S., Zhu, P.,Kramer, O. H., Schimpf, A., Giavara, S., Sleeman, J. P., Lo Coco, F.,Nervi, C., Pelicci, P. G., and Heinzel, T. Valproic acid defines a novelclass of HDAC inhibitors inducing differentiation of transformed cells.EMBO J. 20: 6969-6978, 2001).

HDAC inhibitors are used not only as tumor suppressive agents, but also,for example, as agents for treating and improving autoimmune diseases,skin diseases, infectious diseases, and such (Darkin-Rattray et al.Proc. Natl. Acad. Sci. USA 93, 13143-13147, 1996), as well as inimproving the efficiency of vector introduction in gene therapy (Dion etal., Virology 231, 201-209, 1997), promoting the expression ofintroduced genes (Chen et al., Proc. Natl. Acad. Sci. USA 94, 5798-5803,1997), and the like. Furthermore, HDAC inhibitors are presumed to haveangiogenesis-inhibiting functions (Kim, M. S., Kwon, H. J., Lee, Y. M.,Baek, J. H., Jang, J. E., Lee, S. W., Moon, E. J., Kim, H. S., Lee, S.K., Chung, H. Y., Kim, C. W., and Kim, K. W. (2001). Histonedeacetylases induce angiogenesis by negative regulation of tumorsuppressor genes. Nature Med. 7, 437-443; Kwon, H. J., Kim, M. S., Kim,M. J., Nakajima, H., and Kim, K. W. (2002). Histone deacetylaseinhibitor FK228 inhibits tumor angiogenesis. Int. J. Cancer 97,290-296).

Ten or more HDAC subtypes exist, and recently, specific HDAC subtypeshave been identified as being closely related to cancers. For example,it has been discovered that acetylation of the tumor suppressor genep53, which plays an extremely important role in suppressingcarcinogenesis, is very important in the functional expression of p53itself (Ito, A., Lai, C. H., Zhao, X., Saito, S., Hamilton, M. H.,Appella, E., and Yao, T. P. (2001). p300/CBP-mediated p53 acetylation iscommonly induced by p53-activating agents and inhibited by MDM2. EMBO J.20, 1331-1340), and HDAC1 and HDAC2 are involved in the inhibition ofp53 function (Juan, L. J., Shia, W. J., Chen, M. H., Yang, W. M., Seto,E., Lin, Y. S., and Wu, C. W. (2000). Histone Deacetylases SpecificallyDown-regulate p53-dependent Gene Activation. J. Biol. Chem. 275,20436-20443). It has also been discovered that proteins PML-RAR andPLZF-RAR, involved in the onset of promyelocytic leukemia (APL), andoncogenes such as Bcl-6, which is involved in the onset of lymphomas,recruit HDAC4 or such via nuclear co-repressors, and suppress expressionof the gene group necessary for normal differentiation, causingcarcinogenesis (Dhordain P., Albagli, O., Lin, R. J., Ansieau, S.,Quief, S., Leutz, A., Kerckaert, J. P., Evans, R. M., and Leprince, D.(1997). Corepressor SMRT binds the BTB/POZ repressing domain of theLAZ3/BCL6 oncoprotein. Proc. Natl. Acad. Sci. USA 94, 10762-10767;Grignani, F., De, M. S., Nervi, C., Tomassoni, L., Gelmetti, V., Cioce,M., Fanelli, M., Ruthardt, M., Ferrara, F. F., Zamir, I., Seiser, C.,Grignani, F., Lazar, M. A., Minucci, S., and Pelicci, P. G. (1998).Fusion proteins of the retinoic acid receptor-alpha recruit histonedeacetylase in promyelocytic leukaemia. Nature 391, 815-818; He, L. Z.,Guidez, F., Tribioli, C., Peruzzi, D., Ruthardt, M., Zelent, A., andPandolfi, P. P. (1998). Distinct interactions of PML-RARalpha andPLZF-RARalpha with co-repressors determine differential responses to RAin APL. Nature Genet. 18, 126-135; Lin, R. J., Nagy, L., Inoue, S.,Shao, W., Miller, W. J., and Evans, R. M. (1998). Role of the histonedeacetylase complex in acute promyelocytic leukaemia. Nature 391,811-814). On the other hand, HDAC subtypes which play a very importantrole in the development and differentiation of normal tissues are knownto exist among those HDAC subtypes with tissue-specific expression(McKinsey, T. A., Zhang, C. L., Lu, J., and Olson, E. N. (2000).Signal-dependent nuclear export of a histone deacetylase regulatesmuscle differentiation. Nature 408, 106-111; Verdel, A., and Khochbin,S. (1999). Identification of a new family of higher eukaryotic histonedeacetylases. Coordinate expression of differentiation-dependentchromatin modifiers. J. Biol. Chem. 274, 2440-2445). In order to avoidinhibition of these HDACs, development of a subtype-specific inhibitoris thought to be necessary.

HDAC6 is an enzyme which is shuttled between the nucleus and thecytoplasm by nucleo-cytoplasmic transport, and which normally locates inthe cytoplasm (Verdel, A., Curtet, S., Brocard, M. -P., Rousseaux, S.,Lemercier, C., Yoshida, M., and Khochbin, S. (2000). Active maintenanceof mHDA2/mHDAC6 histone-deacetylase in the cytoplasm. Curr. Biol. 10,747-749). HDAC6 is highly expressed in the testes, and is presumed torelate to the differentiation of normal tissues. Furthermore, HDAC6 isknown to be associated with microtubule deacetylation, and to controlmicrotubule stability (Matsuyama, A., Shimazu, T., Sumida, Y., Saito,A., Yoshimatsu, Y., Seigneurin-Berny, D., Osada, H., Komatsu, Y.,Nishino, N., Khochbin, S., Horinouchi, S., and Yoshida, M. (2002). Invivo destabilization of dynamic microtubules by HDAC6-mediateddeacetylation. EMBO J. 21, 6820-6831). HDAC6 is also a deacetylationenzyme bonded to a microtubule and affecting cell mobility (Hubbert, C.,Guardiola, A., Shao, R., Kawaguchi, Y., Ito, A., Nixon, A., Yoshida, M.,Wang, X. -F., and Yao, T. -P. (2002). HDAC6 is a microtubule-associateddeacetylase. Nature 417, 455-458). Accordingly, HDAC6 inhibitors may bemetastasis-suppressing agents. TSA inhibits each HDAC subtype to aboutthe same degree. However, HDAC6 cannot be inhibited by trapoxinscomprising cyclic tetrapeptide structure and epoxyketone as activegroups (Furumai, R., Komatsu, Y., Nishino, N., Khochbin, S., Yoshida,M., and Horinouchi, S. Potent histone deacetylase inhibitors built fromtrichostatin A and cyclic tetrapeptide antibiotics including trapoxin.Proc. Natl. Acad. Sci. USA 98: 87-92, 2001). Based on the information onthe three-dimensional structure of the enzyme, trapoxins are assumed toexert poor binding properties to HDAC6 due to the structure of itscyclic tetrapeptide moiety that interacts with the weakly conservedoutward surface of the enzyme active center. This implies that alteringthe cyclic tetrapeptide portion may result in inhibitors that areselective for a variety of HDAC.

TSA shows inhibition activity due to the coordination of its hydroxamicacid group with zinc in the HDAC active pocket (Finnin, M. S., Donigian,J. R., Cohen, A., Richon, V. M., Rifkind, R. A., Marks, P. A., Breslow,R., and Pavletich, N. P. Structures of a histone deacetylase homologuebound to the TSA and SAHA inhibitors. Nature 401: 188-193, 1999).Examples of known HDAC inhibitors comprising hydroxamic acid areOxamflatin (Kim, Y. B., Lee, K. -H., Sugita, K., Yoshida, M., andHorinouchi, S. Oxamflatin is a novel antitumor compound that inhibitsmammalian histone deacetylase. Oncogene 18: 2461-2470, 1999) and CHAP(Furumai, R., Komatsu, Y., Nishino, N., Khochbin, S., Yoshida, M., andHorinouchi, S. Potent histone deacetylase inhibitors built fromtrichostatin A and cyclic tetrapeptide antibiotics including trapoxin.Proc. Natl. Acad. Sci. USA 98: 87-92, 2001., Komatsu, Y., Tomizaki, K.-y., Tsukamoto, M., Kato, T., Nishino, N., Sato, S., Yamori, T., Tsuruo,T., Furumai, R., Yoshida, M., Horinouchi, S., and Hayashi, H. CyclicHydroxamic-acid-containing Peptide 31, a potent synthetic histonedeacetylase inhibitor with antitumor activity. Cancer Res. 61:4459-4466, 2001). However, since TSA is instable in blood and has astrong hydroxamic acid chelating function, it chelates with otheressential metal ions, and therefore, HDAC inhibitors comprisinghydroxamic acid have not actually been used as antitumor agents to date.Meanwhile, thiol groups produced by the reduction of FK228 disulfidebonds have recently been shown to serve as active groups to becoordinated with zinc in the HDAC active pocket, inhibiting HDAC. Thus,FK228 is a prodrug that is activated when reduced by cellular reducingactivity (Furumai, R., Matsuyama, A., Kobashi, N., Lee, K. -H.,Nishiyama, M., Nakajima, H., Tanaka, A., Komatsu, Y., Nishino, N.,Yoshida, M., and Horinouchi, S. (2002). FK228 (depsipeptide) as anatural prodrug that inhibits class I histone deacetylases. Cancer Res.62, 4916-4921).

Furthermore, a number of HDAC inhibitors comprising cyclic tetrapeptidestructures and epoxyketones as active groups have been isolated fromnatural environments. On the basis of such findings, the cyclictetrapeptide structure is suggested to be useful in enzymeidentification (as described above, Yoshida, et al., 1995), however,from various viewpoints such as stability, existing inhibitors have notadvanced to the level of being satisfactorily qualified aspharmaceutical products. Therefore, production of pharmaceutical agentsin which these problematic points have been resolved is stronglyanticipated.

DISCLOSURE OF THE INVENTION

The present inventors aim to provide novel HDAC inhibitors comprising acyclic tetrapeptide structure, and methods for producing the same.

In consideration of the above-mentioned objectives, the inventors of thepresent invention synthesized compounds comprising cyclic tetrapeptidestructures that comprise thiol groups and their disulfide bonds, andthen analyzed the HDAC inhibition activity of these compounds. As aresult, it was found that compounds comprising disulfide bonds did notexhibit very high HDAC inhibition activity against enzymes in vitro.However, when converted into thiols by coexisting with the reducingagent dithiothreitol, they showed strong HDAC inhibition activity. Onthe other hand, the intracellular level of disulfide activity wasobserved to be as high as that of TSA and thiols. Accordingly,disulfides were shown to be useful as prodrugs for HDAC inhibitors, inwhich the disulfide bonds are cleaved by intracellular reduction afterbeing taken up into cells, inducing strong activity. Furthermore, thecompounds were found to be more stabile in the serum when the thiolgroups were protected in such a manner, and it was discovered that bybinding the protection groups (-SX) with various functional compounds,the compounds could bind to compounds with desired activities, otherthan HDAC inhibitors.

The invention relates to HDAC inhibitors and methods for producing thesame, and specifically provides the following [1] to [9]:

[1] A compound represented by the following formula (1):

[wherein, R₁₁, R₂₁, R₃₁, and R₄₁ independently denote hydrogen ormethyl; R₂₂, R₂₃, R₃₂, R₃₃, R₄₂, and R₄₃ independently denote ahydrogen, a linear alkyl with one to six carbon atoms, a linear alkylwith one to six carbon atoms to which a non-aromatic cyclic alkyl groupor substituted or unsubstituted aromatic ring, a non-aromatic cyclicalkyl, or a non-aromatic cyclic alkyl group to which a non-aromaticcyclic alkyl group or a substituted or unsubstituted aromatic ring isbound; the pairs of R₂₁ and R₂₂, R₂₂ and R₂₃, R₃₁ and R₃₂, R₃₂ and R₃₃,R₄₁, and R₄₂, and R₄₂ and R₄₃ independently denote acyclic structureswithout binding or cyclic structures by binding through a linearalkylene group with a one- to five-carbon main chain, a linear alkylenegroup with a one- to five-carbon main chain comprising a branched chainwith a one to six carbons, or a linear alkylene group with a one- tofive-carbon main chain comprising a ring structure of one to sixcarbons; X denotes hydrogen, a structure identical to that shown to theleft of X, a substituted or unsubstituted alkyl or aryl group in anystructure comprising a sulfur atom capable of binding with the sulfuratom in formula (1) through a disulfide bond, or a sulfur atom bindingwith the sulfur atom bonded to the terminus of R₂₂, R₂₃, R₃₂, R₃₃, R₄₂,or R₄₃, and located to the left of X, via an intramolecular disulfidebond].

[2] A histone deacetylase inhibitor that comprises the compound of [1]as an active ingredient.

[3] An apoptosis inducing agent that comprises the compound of [1] as anactive ingredient.

[4] A differentiation-inducing agent that comprises the compound of [1]as an active ingredient.

[5] An angiogenesis inhibitor that comprises the compound of [1] as anactive ingredient.

[6] An anti-metastatic agent comprising the compound of [1] as an activeingredient.

[7] A pharmaceutical agent for treating or preventing a disease causedby histone deacetylase 1 or 4, comprising the compound of [1] as anactive ingredient.

[8] The pharmaceutical agent of [7], wherein the disease caused byhistone deacetylase 1 or 4 is cancer, autoimmune disease, skin disease,or infectious disease.

[9] A method for producing the compound of [1], which comprises thesteps of:reacting a compound represented by formula (2)

(wherein, n is same as that defined in formula (1); Hal denotes ahalogen atom selected from a chlorine atom, bromine atom, or iodineatom, or an allyl or alkylsulfoxy group useful for a free group; P₂denotes a protection group for an amino group);with a compound represented by formula (3).

(wherein R₁₁, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, R₃₃, R₄₁, R₄₂, and R₄₃ are sameas defined in formula (1); P₂ denotes a protection group for a carboxylgroup);in the presence of a peptide-bonding agent to obtain a compoundrepresented by formula (4)

(wherein n, R₁₁, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, R₃₃, R₄₁, R₄₂, R₄₃, P₁, P₂,and Hal are the same as defined above);subjecting the compound represented by formula (4) to catalytichydrogenation, acid treatment, or hydrolysis to remove P₁ and P₂;and then subjecting to cyclization in the presence of a peptide-bondingagent to obtain a compound represented by formula (5)

(wherein n, R₁₁, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, R₃₃, R₄₁, R₄₂, R₄₃, P₁, P₂,and Hal are the same as defined above);or reacting a compound represented by formula (6)

(wherein R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, R₃₃, R₄₁, R₄₂, R43, and P₁ are thesame as defined above);with a compound represented by formula (7)

(wherein n, R₁₁, P₂, and Hal are the same as defined above);in the presence of a peptide-bonding agent to obtain a compoundrepresented by formula (8)

(wherein n, R₁₁, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, R₃₃, R₄₁, R₄₂, R₄₃, P₁, P₂,and Hal are the same as defined above);subjecting the compound represented by formula (8) to catalytichydrogenation, acid treatment, fluoride anion treatment, or hydrolysisto remove P₁ and P₂;and then subjecting to cyclization in the presence of a peptide-bondingagent to obtain the compound represented by formula (5);following, for both process, the steps of:reacting the compound represented by formula (5) with a reagentcomprising sulfur atoms to obtain a compound represented by formula (9)

(wherein n, R₁₁, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, R₃₃, R₄₁, R₄₂, and R₄₃ are thesame as defined above; P₃ denotes a protection group for sulfohydrylgroup); and thentreating the compound represented by formula (9) with an oxidizing agentas well as ammonia or another amine.

Hereinafter, modes for carrying out the present invention will bespecifically described with reference to drawings.

The compounds of the present invention can be defined by theabove-mentioned formula (1). Such compounds can be used as the HDACinhibitors.

In the above-mentioned formula (1), R₁₁, R₂₁, R₃₁, and R₄₁ may eachindependently be hydrogen or methyl. R₂₂, R₂₃, R₃₂, R₃₃, R₄₂, and R₄₃may each independently be hydrogen, a linear alkyl with one to sixcarbon atoms, or a non-aromatic cyclic alkyl group, in which the linearalkyl group with one to six carbon atoms and the non-aromatic cyclicalkyl group may bind with a non-aromatic cyclic alkyl group, orsubstituted or unsubstituted aromatic ring. Pairs of R₂₁ and R₂₂, R₂₂and R₂₃, R₃₁ and R₃₂, R₃₂ and R₃₃, R₄₁ and R₄₂, and R₄₂ and R₄₃ can eachindependently be in an acyclic structure without binding, or may bindthrough a linear alkylene group with a one- to five-carbon main chain, alinear alkylene group with a one- to five-carbon main chain comprising abranched chain with one to six carbons, or a linear alkylene group witha one- to five-carbon main chain comprising a ring structure of one tosix carbons. Since the cyclic tetrapeptide structure portion is thoughtto function as a cap to seal a pocket of HDAC, this cap structure can bearbitrarily selected from the above-mentioned linear alkyl with one tosix carbon atoms, aromatic cyclic alkyl, and aromatic groups that cansubstitute for them.

Furthermore, hydrogen can be used as X in formula (1) to directly form athiol group with a neighboring sulfur atom that exhibits HDAC inhibitionactivity. However, if the thiol group formed by using a hydrogen as X isexposed, the resulting compound becomes unstable in vivo. Therefore, ifX is a hydrogen, the present compounds are preferably combined with ameans for their stable delivery to a desired site, such as a drugdelivery system. In order to enhance the stability of thiol groupscomprising HDAC inhibition activity, it is preferable that X is asubstituent group which is metabolized in vivo and harmless in theliving body. This kind of substituent group is preferably a groupcomprising a sulfur atom capable of forming a disulfide bond with thesulfur atom next to the X, and can be a group that itself shows someefficacy, and can also be a group that functions simply as a protectivegroup. Such a substituent group comprising a sulfur atom can be:

a structure identical to that shown to the left of X; an alkyl group oraryl group in any structure comprising a sulfur atom capable of bindingvia a disulfide bond with the sulfur atom in the above-mentioned formula(1); or a sulfur atom binding with the sulfur atom bonded to theterminus of the above-mentioned R₂₂, R₂₃, R₃₂, R₃₃, R₄₂, or R₄₃ andlocated to the left of X via an intra-molecular disulfide bond. In thiscase, if the substituent group has the same structure as that to theleft of X, resulting in a dimer structure, the disulfide bond is cut byin vivo metabolism to isolate an HDAC inhibitor comprising the activityof two molecules. Furthermore, any alkyl or aryl comprising a sulfuratom may have further substituted groups, or may be a structure capableof exhibiting an effect identical to or different from that of the HDACinhibitor.

Examples of the SS-hybrid X atom groups of the present invention arealkylmercaptans such as methylmercaptan, benzylmercaptan andcyclohexylmercaptan, and aromatic mercaptans such as thiophenol andmercaptopyridine, as well as alkylmercaptans and allylmercaptans inwhich a portion of the atom groups in the structure of naturalphysiologically active substances, such as 5-azadeoxycytidine andretinoic acid, are substituted with thiol groups. Preferable examplesare methylmercaptan, ethylmercaptan, mercaptoethanol, cysteamine,cysteine, thiophenol, 2-mercaptopyridine, 4-mercaptopyridine,5′-mercapto-5-azadoxycytidine, 3′-mercapto-5-azadeoxycytidine, andthioretinol.

Furthermore, in the present invention, the ring n in formula (1) is notlimited as long as it has HDAC inhibition activity and, for example, nis preferably 4 to 7, more preferably 5. The carbon chain comprising ncarbon atoms, from the cyclic tetrapeptide structure to the sulfur atom,is supposed to enter the active HDAC pocket, and inhibit HDAC bycontacting the active thiol group at the carbon chain end with the zincmolecule in the HDAC pocket.

Typical examples of the compounds of the present invention are shown inFIGS. 1 to 3, but are not limited to these compounds.

Hereinafter, methods of producing the compounds of the present inventionwill be described. The compounds of this embodiment can be produced from2-amino-n-haloalkanoic acid, as shown below. Since R₁₁, R₂₁, R₂₂, R₂₃,R₃₁, R₃₂, R₃₃, R₄₁, R₄₂, R₄₃, and n are defined according to the abovedescriptions, their descriptions are omitted.

The first embodiment of methods of production for the compounds of thepresent invention is a method that uses as raw material a compound offormula (2), in which protection group P₁ is linked to the amino groupof 2-amino-n-haloalkanoic acid. Specifically, a compound defined by thefollowing formula (2)

is reacted with a compound defined by the following formula (3)

in the presence of a peptide-bonding agent to obtain a compound definedby the following formula (4).

In the above-described formulae, Hal can be a halogen atom selected fromany one of a chlorine atom, a bromine atom, or an iodine atom, or anallyl or alkylsulfoxy group that can also be a leaving group. P₂ is aprotection group for an amino group.

Next, the compound defined by the above-mentioned formula (4) issubjected to catalytic hydrogenation, acid treatment, or hydrolysis forremoving P₁ and P₂, and then to cyclization in the presence of apeptide-bonding agent, to obtain a compound defined by formula (5):

Next, the compound defined by formula (5) is reacted with a reagentcomprising a sulfur atom to obtain a compound defined by formula (9):

The compound defined by formula (9) is then treated with an oxidizingagent as well as ammonia or another amine to obtain a (dimer- orhybrid-type) prodrug compound comprising a disulfide bond. In formula(9), P₃ denotes a protection group for the sulfohydryl group. Treatmentwith a reducing agent or an enzyme capable of digesting disulfide bondsmay be carried out to isolate active thiol-type compounds.

The second embodiment of the production methods of the present inventionis a production method that uses as raw material a compound defined bythe following formula (7), in which a protection group P₂ is linked tothe carboxyl group of 2-amino-n-haloalkanoic acid. Specifically, acompound defined by formula (6)

is reacted with a compound defined by formula (7)

in the presence of peptide-bonding agent, to obtain a compound definedby the following formula (8):

The compound defined by formula (8) is then subjected to catalytichydrogenation, acid treatment, fluoride anion treatment, or hydrolysisto remove P₁ and P₂, and then subjected to cyclization in the presenceof a peptide-bonding agent, to obtain a compound defined by formula (5).Next, the compound defined by formula (5) is reacted with asulfur-atom-comprising reagent to obtain a compound defined by formula(9):

The compound defined by formula (9) is then treated with an oxidizingagent as well as ammonia or another amine to obtain a (dimer- orhybrid-type) prodrug compound comprising a disulfide bond. As for theabove-mentioned first embodiment, active thiol-type compounds may beisolated by treatment with a reducing agent or an enzyme capable ofdigesting disulfide bonds.

HDAC-inhibiting compounds are known to induce differentiation of cancercells, leukemia cells, and neural cells, to induce apoptosis, andsuppress cancer cell metastasis (Yoshida, M., Nomura, S., and Beppu, T.Effects of trichostatins on differentiation of murine erythroleukemiacells. Cancer Res. 47: 3688-3691, 1987; Hoshikawa, Y., Kijima, M.,Yoshida, M., and Beppu, T. Expression of differentiation-related markersin teratocarcinoma cells via histone hyperacetylation by trichostatin A.Agric. Biol. Chem. 55: 1491-1495, 1991; Minucci, S., Horn, V.,Bhattacharyya, N., Russanova, V., Ogryzko, V. V., Gabriele, L., Howard,B. H., and Ozato, K. A histone deacetylase inhibitor potentiatesretinoid receptor action in embryonal carcinoma cells. Proc. Natl. Acad.Sci. USA 94: 11295-11300, 1997; Inokoshi, J., Katagiri, M., Arima, S.,Tanaka, H., Hayashi, M., Kim, Y. B., Furumai, R., Yoshida, M.,Horinouchi, S., and Omura, S. (1999). Neuronal differentiation of Neuro2a cells by inhibitors of cell progression, trichostatin A andbutyrolactone I. Biochem. Biophys. Res. Commun. 256, 372-376; Wang, J.,Saunthararajah, Y., Redner, R. L., and Liu, J. M. Inhibitors of histonedeacetylase relieve ETO-mediated repression and induce differentiationof AML1-ETO leukemia cells. Cancer Res. 59: 2766-2769, 1999; Munster, P.N., Troso-Sandoval, T., Rosen, N., Rifkind, R., Marks, P. A., andRichon, V. M. The histone deacetylase inhibitor suberoylanilidehydroxamic acid induces differentiation of human breast cancer cells.Cancer Res. 61: 8492-8497, 2001; Ferrara, F. F., Fazi, F., Bianchini,A., Padula, F., Gelmetti, V., Minucci, S., Mancini, M., Pelicci, P. G.,Lo Coco, F., and Nervi, C. Histone deacetylase-targeted treatmentrestores retinoic acid signaling and differentiation in acute myeloidleukemia. Cancer Res. 61: 2-7, 2001; Gottlicher, M., Minucci, S., Zhu,P., Kramer, O. H., Schimpf, A., Giavara, S., Sleeman, J. P., Lo Coco,F., Nervi, C., Pelicci, P. G., and Heinzel, T. Valproic acid defines anovel class of HDAC inhibitors inducing differentiation of transformedcells. EMBO J. 20: 6969-6978, 2001). Accordingly, the compounds of thepresent invention can be utilized as apoptosis-inducing agents,differentiation-inducing agents, and cancer-metastasis-suppressingagents.

Also, the compounds inhibiting HDAC are expected to inhibit angiogenesis(Kim, M. S., Kwon, H. J., Lee, Y. M., Baek, J. H., Jang, J. E., Lee, S.W., Moon, E. J., Kim, H. S., Lee, S. K., Chung, H. Y., Kim, C. W., andKim, K. W. (2001). Histone deacetylases induce angiogenesis by negativeregulation of tumor suppressor genes. Nature Med. 7, 437-443; Kwon, H.J., Kim, M. S., Kim, M. J., Nakajima, H., and Kim, K. W. (2002). Histonedeacetylase inhibitor FK228 inhibits tumor angiogenesis. Int. J. Cancer97, 290-296). Thus, the compounds of the present invention can be alsoutilized as angiogenesis inhibitors.

Among various HDACs, the compounds of the present invention exhibit astrong inhibitive activity specific to HDAC1 and HDAC4. Therefore, thecompounds of the present invention are useful as pharmaceutical agentsfor treating or preventing diseases caused by HDAC1 and HDAC4. Examplesof such diseases besides cancer include autoimmune diseases, skindiseases, and infectious diseases associated with HDAC1 and HDAC4.Furthermore, the compounds of the present invention may be applied notonly to pharmaceutical agents for treating or preventing theabove-mentioned diseases, but also to gene therapy adjuvants oraccelerating agents that improve the efficiency of vector introduction,promote the expression of introduced genes, and the like.

The compounds of the present invention may also be used in combinationwith retinoic acids and DNA methylation inhibitors. The invention alsoprovides such concomitant agents.

When formulating the compounds of the present invention, fillers,extenders, binders, moisturizing agents, disintegrators, surfactants,diluents such as lubricants, and vehicles may be used as necessary.Furthermore, coloring agents, preservatives, aromatics, flavors,sweeteners, and other pharmaceuticals may be added to the pharmaceuticalformulations. The form of each type of pharmaceutical formulation may beselected in line with its therapeutic or preventative purpose. The formmay be, for example, a tablet, pill, powder, solution, suspension,emulsion, granule, capsule, injection, and suppository.

Examples of additives to be added to tablets and capsules includebinders such as gelatin, corn starch, tragacanth gum, and acacia;vehicles such as crystalline cellulose; swelling agents such as cornstarch, gelatin, and alginic acid; lubricants such as magnesiumstearate; sweeteners such as sucrose, lactose, and saccharine; andaromatics such as peppermint, Gaultheria adenothrix oil, and cherry. Inthe case where the unit dosage form is a capsule, a liquid carrier suchas oil or fat can be added in addition to the above-mentioned materials.

As an aqueous solution for injection, an isotonic solution of, forexample, D-sorbitol, D-mannose, D-mannitol, or sodium chloridecomprising saline, glucose, and other adjuvants may also be used asnecessary in combination with a proper dissolution-assisting agent, suchas an alcohol, specifically, ethanol, a polyalcohol such as propyleneglycol and polyethylene glycol, or a nonionic surfactant such aspolysorbate 80™ and HCO-50.

Examples of an oleaginous solution are sesame oil and soybean oil, whichcan be used, as necessary, in combination with a dissolution-assistingagent such as benzyl benzoate and benzyl alcohol. Furthermore, mixingwith a buffer such as phosphate buffer solution or sodium acetate buffersolution; a soothing agent such as procaine hydrochloride; a stabilizersuch as benzyl alcohol and phenol; or an antioxidant is also acceptable.The formulated injection is generally filled into suitable ampules.

Formulations may be administered to patients orally or parenterally.Examples of a parenteral dosage form, include injection as well astransnasal, transpulmonal, and transdermal administration. Systemic orlocal administration can be carried out using an injection dosage form,such as intravenous injection, intramuscular injection, intraperitonealinjection, and subcutaneous injection. Furthermore, intranasal,transbronchial, intramuscular, subcutaneous, or oral administration mayalso be carried out by methods known to those skilled in the art.

For parenteral administration, the unit dosage of the compounds of thepresent invention depends on the subjects to be administrated, thetarget organs, symptoms, and the manner of administration. For example,it is preferable that injections are administered intravenously toadults (60 kg body weight) at a dosage of about 0.01 to 30 mg per day,preferably about 0.1 to 20 mg per day, and more preferably about 0.1 to10 mg per day. When administering to other kind of animals, dosage canbe converted per 60 kg body weight, or per unit of body surface area.

For oral administration, the unit dosage of the compounds of the presentinvention depends on the subjects to be administrated, the targetorgans, symptoms, and manner of administration, and is preferably, forexample, about 100 μg to 20 mg per day for an adult (60 kg body weight).

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 shows a list of the structure of LDLD or LDLL isomers of SCOPwith five carbon chains until the active thiol group.

FIG. 2 shows the structures of SCOPs with four to seven carbon chainsuntil the active thiol group. Note that SCOP 152 (C5) is the same hereas in FIG. 1.

FIG. 3 shows the structures of homodimer-type SCOPs. The SCOP numbersare twice the number of monomers.

FIG. 4 shows the structures of hybrid-type SCOPs, obtained by bonding avariety of compounds to SCOP 152.

FIG. 5 shows the steric conformation of natural Cyl-1 and Cyl-2.

FIG. 6 shows photographs of the results of measuring intracellularhistone acetylation level by Western blot analysis using anti-acetylatedlysine antibodies.

FIG. 7 shows the results of evaluating the stability of SCOP 152, SCOP304, and SCOP 402 in serum.

FIG. 8 shows photographs of the results of evaluating the stability ofSCOP 152, SCOP 304, and SCOP 402 on a cellular level.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, FIGS. 1 and 2 show the entire flow of the process ofsynthesizing the compounds shown in these examples. Each synthesis stepis described in detail below, with H-L-Ab7-OH as the starting material.In the following, 2-amino-7-bromoheptanoic acid is abbreviated to “Ab7”; 2-amino-7-acetyltioheptanoic acid is “Am7(Ac)”;2-amino-7-mercaptoheptanioc acid is “Am7”; sulfide of2-amino-7-mercaptoheptanioc acid is “Am7(-)”;2-amino-8.9-dimercapto(S⁹-2′-nitro-N,N′-dimethyl benzamide) is“Am7(Ell)”; 2-amino-8,9-dimercapto-11-hydroxyundecanoic acid is“Am7(SMEt)”; 2-amino-8.9-dimercapto(S⁹-2′-pyridyl) nonanoic acid is“Am7(S2Py)”; 2-amino-8.9-dimercapto(S⁹-4′-pyridyl) nonanoic acid is“Am7(S4Py)”; and 2-amino-8,9-dimercaptodecanoic acid is “Am7(SMe)”. Inaddition, sulfur-containing cyclic peptides, which are synthesizedcompounds, are abbreviated to “SCOP”.

EXAMPLE 1 Synthesis of Boc-L-Ab7-OH

H-L-Ab7-OH (7.3 g, 32.4 mmol) was dissolved in water:dioxane=1:1solution (30 ml, v/v). While cooling on ice, (Boc)₂O (7.68 g, 35.6 mmol)and triethylamine (6.72 ml, 48.6 mmol) were added to the mixture, whichwas then stirred for five hours. After the reaction solution wasevaporated, the residue was washed with ether. The aqueous phase wasacidified using citric acid and reverse-extracted with ethyl acetate.The extract was dried over MgSO₄ and ethyl acetate was then removed byevaporation. After vacuum drying, the oily title compound (10.4 g, 32.4mmol, 100% yield) was obtained.

EXAMPLE 2 Synthesis of Boc-L-Ab7-NHMe

While cooling on ice, triethylamine (0.17 ml, 1.2 mmol) and DCC (247 mg,1.2 mmol) were added to 3 ml of DMF containing Boc-L-Ab7-OH (326 mg, 1.0mmol), monomethylamine hydrochloride (81 mg, 1.2 mmol), and HOBt.H₂O(184 mg, 1.2 mmol). After stirring for 15 hours, the DMF was removed byevaporation. The residue was dissolved in ethyl acetate and successivelywashed with an aqueous 10% citric acid solution, an aqueous 4% sodiumbicarbonate solution, and saturated saline. This was then dried overMgSO₄ and concentrated. The resulting oily substance was purified byflash silica gel chromatography (3.6×15 cm, chloroform) and solidifiedby adding ether/petroleum ether (1:10) to obtain the white powder of thetitle compound (250 mg, 0.74 mmol, 74% yield). TLC: Rf=0.58(CHCl₃/MeOH=9/1).

EXAMPLE 3 Synthesis of Boc-L-Am7(Ac)-NHMe

Potassium thioacetate (64 mg, 0.56 mmol) was added to DMF (2 ml)containing Boc-Ab7-NHMe (125 mg, 0.37 mmol) and reacted for 3 hours.After DMF was removed by evaporation, the residue was dissolved in ethylacetate and successively washed with an aqueous 10% citric acid solutionand saturated saline. The product was dried over MgSO₄, concentrated,and solidified by adding ether/petroleum ether (1:10) to obtain thewhite powder of the title compound (120 mg, 0.36 mmol, 97% yield). TLC:Rf=0.57 (CHCl₃/MeOH=9/1).

EXAMPLE 4 Synthesis of Boc-L-Am7(-)-NHMe SS-dimer

Methanolic ammonia (20 eq.) was added to DMF (0.5 ml) containingBoc-Am7(Ac)-NHMe (60 mg, 0.18 mmol) and stirred for 24 hours. Afterconcentrating the reaction solution, the SS-dimer produced was purifiedby flash silica gel chromatography (1.5×30 cm, 1% methanol/chloroform)to obtain the white powder of the title compound (43 mg, 0.11 mmol, 61%yield). HPLC retention time: 8.5 min; HRMS (FAB, dithiodiethanol),579.3293 [M+H], C₂₆H₅₁O₆N₄S₂ (579.3250).

EXAMPLE 5 Synthesis of Boc-L-Am7(S4Py)-NHMe

4,4′-dithiodipyridine (79 mg, 0.36 mmol) and methanolic ammonia (20 eq.)were added to DMF (0.5 ml) containing Boc-Am7(Ac)-NHMe (60 mg, 0.18mmol), and stirred for 5 hours. After the reaction solution wasconcentrated, the resulting oily product was purified by flash silicagel chromatography (1.5×30 cm, chloroform) and freeze-dried to obtainthe title compound (43 mg, 0.11 mmol, 61% yield). HPLC retention time:5.6 min; HRMS (FAB, dithiodiethanol), 400.1766 [M+H], C₁₈H₂₉O₃N₃S₂(400.1729).

EXAMPLE 6 Synthesis of Boc-L-Ab7-OBzl

Boc-L-Ab7-OH (4.05 g, 12.5 mmol) was dissolved in DCM (20 ml). Whilecooling on ice, benzyl alcohol (1.55 ml, 15.0 mmol),4-dimethylaminopyridine (153 mg, 1.25 mmol), and DCC (3.09 g, 15.0 mmol)were added to the mixture, which was then stirred for 8 hours. After thereaction solution was evaporated, the residue was dissolved in ethylacetate and successively washed with an aqueous 10% citric acidsolution, an aqueous 4% sodium bicarbonate solution, and saturatedsaline. This was then dried over MgSO₄, concentrated, and the resultingoily substance was purified by flash silica gel chromatography (5×20 cm,20% ethyl acetate/hexane) to obtain the oily title compound (4.29 g,10.4 mmol, 83% yield). TLC: Rf=0.49 (ethyl acetate/hexane=1/4).

EXAMPLE 7 Synthesis of Boc-L-Ile-L-Pro-OBzl

While cooling on ice, Boc-L-Pro-OH (1.08 g, 5.0 mmol) and benzyl bromide(0.893 ml, 75 mmol) were reacted in DMF (10 ml) in the presence oftriethylamine (10.5 ml, 75 mmol) to obtain Boc-L-Pro-OBzl as an oilyproduct. This product was reacted under ice-cooling with 2 N HCl/dioxane(5 eq.) for three hours to obtain H-L-Pro-OBzl-HCl.

While cooling on ice, DCC (1.24 g, 6.0 mmol) and triethylamine (0.70 ml,4.0 mmol) were added to DMF (10 ml) containing Boc-L-Ile-OH.1/2 H₂O(1.39 g, 6.0 mmol), H-D-Pro-OBzl-HCl (956 mg, 4.0 mmol), and HOBt.H₂O(613 mg, 4.0 mmol). After stirring for 8 hours, DMF was removed byevaporation, the residue was dissolved in ethyl acetate, and thensuccessively washed with an aqueous 10% citric acid solution, an aqueous4% sodium bicarbonate solution, and saturated saline. After drying overMgSO₄ and concentrating, the resulting oily substance was purified byflash silica gel chromatography (4×30 cm, 1% methanol/chloroform). toobtain the oily title compound (1.63 g, 3.38 mmol, 85% yield). TLC:Rf=(CHCl₃/MeOH=9/1)

EXAMPLE 8 Synthesis of Boc-D-Tyr(Me)-L-Ile-L-Pro-OBzl

Boc-L-Ile-L-Pro-OBzl (1.63 g, 3.38 mmol) was dissolved in TFA (5 ml) andleft with standing on ice for 30 minutes. On completion of the reaction,TFA was removed by evaporation, and the residue was vacuum-dried toobtain H-L-Ile-L-Pro-OBzl-TFA. The compound was dissolved in DMF (8 ml),and Boc-D-Tyr(Me)-OH (1.50 g, 5.07 mmol) was then added. HBTU (1.92 g,5.07 mmol), HOBt.H₂O (518 mg, 3.38 mmol), and triethylamine (2.37 ml,16.9 mmol) were further added and stirred for three hours underice-cooling. The reaction solution was concentrated, dissolved in ethylacetate, and successively washed with an aqueous 10% citric acidsolution, an aqueous 4% sodium bicarbonate solution, and saturatedsaline. The product was dried over MgSO₄ and concentrated, and theresulting foam substance was purified by flash silica gel chromatography(4×30 cm, 1% methanol/chloroform) to obtain the foam title compound(1.44 g, 2.42 mmol, 72% yield). TLC: Rf=(CHCl₃/MeOH=9/1).

EXAMPLE 9 Synthesis of Boc-D-Tyr(Me)-L-Ile-L-Pro-L-Ab7-OBzl

Boc-D-Tyr(Me)-L-Ile-L-Pro-OBzl (1.44 g, 2.42 mmol) was dissolved inmethanol (12 ml) and subjected to catalytic hydrogenation in thepresence of the 5% Pd-C (150 mg). After five hours, the catalyst Pd-Cwas filtered and the reaction solution was removed by evaporation toobtain Boc-D-Tyr(Me)-L-Ile-L-Pro-OH-TFA.

Boc-L-Ab7-OBzl (1.29 g, 3.12 mmol) was dissolved in TFA (10 ml) and leftwith standing on ice for 30 minutes. On completion of the reaction, TFAwas removed by evaporation, and the residue was vacuum-dried to obtainH-L-Ab7-OH-TFA. The compound was dissolved in DMF (16 ml), andBoc-D-Tyr(Me)-L-Ile-L-Pro-OH (1.21 g, 2.40 mmol) was then added. HBTU(1.18 g, 3.12 mmol), HOBt.H₂O (368 mg, 2.40 mmol), and triethylamine(1.34 ml, 9.6 mmol) were further added and stirred for 3 hours underice-cooling. The reaction solution was concentrated, dissolved in ethylacetate, and successively washed with 10% citric acid, an aqueous 4%sodium bicarbonate solution, and saturated saline. The product was driedover MgSO₄ and concentrated, and the resulting foam substance waspurified by flash silica gel chromatography (4×30 cm, 2%methanol/chloroform) to obtain the foam title compound (1.20 g, 1.47mmol, 61% yield). TLC: Rf=(CHCl₃/MeOH=9/1).

EXAMPLE 10 Synthesis of H-D-Tyr(Me)-L-Ile-L-Pro-L-Ab7-OH.TFA

Boc-D-Tyr(Me)-L-Ile-L-Pro-L-Ab7-OBzl (1.20 g, 1.47 mmol) was dissolvedin methanol (7.5 ml) and subjected to catalytic hydrogenation in thepresence of the catalyst Pd-C (130 mg). After 5 hours, the catalyst Pd-Cwas filtered and the reaction solution was removed by evaporation toobtain Boc-D-Tyr(Me)-L-Ile-L-Pro-L-Ab7-OH. This compound was dissolvedin TFA (5 ml) and left standing on ice for 30 minutes. After thereaction solution was removed by evaporation, ether/petroleum ether(1:10) was added to the residue for solidification. This was thenvacuum-dried to obtain the title compound (770 mg, 1.02 mmol, 69%yield).

EXAMPLE 11 Synthesis of cyclo(-L-Ab7-D-Tyr(Me)-L-Ile-L-Pro-)

H-D-Tyr(Me)-L-Ile-L-Pro-L-Ab7-OH.TFA (770 mg, 1.02 mmol), HATU (388 mg,1.53 mmol), and DIEA (0.71 ml) were divided into five aliquots. DMF(1000 ml) was added to each aliquot every 30 minutes, and cyclizationreaction was carried out. After 2 hours, the solvent was removed byevaporation. The residue was dissolved in ethyl acetate, successivelywashed with an aqueous 10% citric acid solution, 4% NaHCO₃, andsaturated saline, and then dried with MgSO₄. Ethyl acetate was removedby evaporation, and the remaining oily substance was purified by flashsilica gel chromatography (4×30 cm, 2% methanol/chloroform) to obtain afoam substance 130 mg (21%). HPLC retention time: 8.20 min; FAB-MS(dithiodiethanol), 593 [M+H], (593.2).

EXAMPLE 12 Synthesis of cyclo(-L-Am7(Ac)-D-Tyr(Me)-L-Ile-L-Pro-)

Potassium thioacetate (9.59 mg, 0.084 mmol) was added to DMF (0.5 ml)containing cyclo(-L-Ab7-D-Tyr(Me)-L-Ile-L-Pro-) (25 mg, 0.042 mmol) andreacted for 3 hours. DMF was removed by evaporation. The residue wasdissolved in ethyl acetate and then successively washed with aqueous 10%citric acid solution, 4% NaHCO₃, and saturated saline. The thioesterproduced similarly after the cyclization reaction was isolated andpurified to obtain 19 mg (76%) of oily product. HPLC retention time:8.20 min; FAB-MS (dithiodiethanol), 589 [M+H], (589.3).

EXAMPLE 13 Synthesis of cyclo(-L-Am7(-)-D-Tyr(Me)-L-Ile-L-Pro-)(SS-dimer: SCOP 296)

Cyclo(-L-Am7(Ac)-D-Tyr(Me)-L-Ile-L-Pro-) (19 mg, 0.0322 mmol) wasdissolved in hot DMF (2 ml) and reacted with methanolic ammonia (10 eq.)to remove the acetyl groups. After the solvent was removed byevaporation, the residue was dissolved in DMF (2 ml), and 1 M I₂(ethanol) (0.04 ml) was added to the solution for oxidization. Theproduced SS-dimer was purified through a Sephadex LH-20 (DMF) column andthen mixed with water to obtain a white powder. The yield was 7.4 mg(42%). HPLC retention time: 14.1 min; HRMS (FAB, dithiodiethanol),1091.5648 [M+H], C₅₆H₈₃O₁₀N₈S₂ (1091.5674).

EXAMPLE 14 Synthesis of Boc-L-Ile-DL-Pip-OBzl

Boc-DL-Pip-OH (2.29 g, 10 mmol) and benzyl bromide (1.79 ml, 15 mmol)were reacted in DMF (20 ml) in the presence of triethylamine (2.1 ml, 15mmol) to obtain Boc-DL-Pip-OBzl as an oily product. This product wasreacted with 2 N HCl/dioxane (5 eq.) for 3 hours to obtainH-DL-Pip-OBzl-HCl.

While cooling on ice, DCC (2.20 g, 10.7 mmol) and triethylamine (1.25ml, 8.9 mmol) were added to DMF (20 ml) containing Boc-L-Ile-OH.1/2 H₂O(2.47 g, 10.7 mmol), H-D-Pro-OBzl-HCl (2.28 g, 8.9 mmol), and HOBt.H₂O(1.36 mg, 8.9 mmol). After stirring for 8 hours, DMF was removed byevaporation, the residue was dissolved in ethyl acetate, and thensuccessively washed with an aqueous 10% citric acid solution, an aqueous4% sodium bicarbonate solution, and saturated saline. After drying overMgSO₄ and concentrating, the resulting oily substance was purified byflash silica gel chromatography (4×30 cm, 1% methanol/chloroform) toobtain the oily title diastereomer mixture (3.33 g, 7.70 mmol, 87%yield). TLC: Rf=(CHCl₃/MeOH=9/1)

EXAMPLE 15 Synthesis of Boc-D-Tyr(Me)-L-Ile-DL-Pip-OBzl

Boc-L-Ile-DL-Pip-OBzl (3.33 g, 7.70 mmol) was dissolved in TFA (10 ml)and left with standing on ice for 30 minutes. On completion of thereaction, TFA was removed by evaporation, and the residue wasvacuum-dried to obtain H-L-Ile-DL-Pip-OBzl.TFA. The compound wasdissolved in DMF (16 ml), and Boc-D-Tyr(Me)-OH (3.41 g, 11.6 mmol) wasthen added. HBTU (4.38 g, 11.6 mmol), HOBt.H₂O (1.18 g, 7.70 mmol), andtriethylamine (7.01 ml, 50.1 mmol) were further added and stirred for 3hours under ice-cooling. The reaction solution was concentrated,dissolved in ethyl acetate, and successively washed with an aqueous 10%citric acid solution, an aqueous 4% sodium bicarbonate solution, andsaturated saline. The product was dried over MgSO₄ and concentrated, andthe resulting foam substance was purified by flash silica gelchromatography (4×30 cm, 1% methanol/chloroform) to obtain the foamtitle diastereomer mixture (3.46 g, 5.67 mmol, 74% yield). TLC:Rf=(CHCl₃/MeOH=9/1).

EXAMPLE 16 Synthesis of Boc-D-Tyr(Me)-L-Ile-DL-Pip-L-Ab7-OBzl

Boc-D-Tyr(Me)-L-Ile-DL-Pip-OBzl (3.46 g, 7.37 mmol) was dissolved inmethanol (30 ml) and subjected to catalytic hydrogenation in thepresence of the 5% Pd-C (230 mg). After 8 hours, the catalyst Pd-C wasfiltered and the reaction solution was evaporated to obtainBoc-D-Tyr(Me)-L-Ile-DL-Pip-OH.

Boc-L-Ab7-OBzl (3.05 g, 3.12 mmol) was dissolved in TFA (5 ml) and leftwith standing on ice for 30 minutes. On completion of the reaction, TFAwas removed by evaporation, and the residue was vacuum-dried to obtainH-L-Ab7-OBzl.TFA. The compound was dissolved in DMF (16 ml), andBoc-D-Tyr(Me)-L-Ile-DL-Pip-OH (2.80 g, 5.39 mmol) was then added. HBTU(2.66 g, 7.01 mmol), HOBt.H₂O (825 mg, 5.39 mmol), and triethylamine(3.02 ml, 21.6 mmol) were further added and stirred for 3 hours underice-cooling. The reaction solution was concentrated, dissolved in ethylacetate, and successively washed with an aqueous 10% citric acidsolution, an aqueous 4% sodium bicarbonate solution, and saturatedsaline. The product was dried over MgSO₄ and concentrated, and theresulting foam substance was purified by flash silica gel chromatography(4×30 cm, 2% methanol/chloroform) to obtain the foam title diastereomermixture (4.07 g, 4.91 mmol, 91% yield). TLC: Rf=(CHCl₃/MeOH=9/1).

EXAMPLE 17 Synthesis of H-D-Tyr(Me)-L-Ile-DL-Pip-L-Ab7-OH.TFA

Boc-D-Tyr(Me)-L-Ile-L-Pro-DL-Pip-OBzl (4.07 g, 4.91 mmol) was dissolvedin methanol (10 ml) and subjected to catalytic hydrogenation in thepresence of the catalyst Pd-C (300 mg). After 8 hours, the catalyst Pd-Cwas filtered and the reaction solution was removed by evaporation toobtain Boc-D-Tyr(Me)-L-Ile-DL-Pip-OH. This compound was dissolved in TFA(10 ml) and left standing on ice for 30 minutes. After the reactionsolution was concentrated by evaporation, ether/petroleum ether (1:10)was added to the residue for solidification. This was then vacuum-driedto obtain the title diastereomer mixture (2.60 g, 3.51 mmol, 72% yield).

EXAMPLE 18 Synthesis of cyclo(-L-Ab7-D-Tyr(Me)-L-Ile-L-Pip-) andcyclo(-L-Ab7-D-Tyr(Me)-L-Ile-D-Pip-)

A linear tetrapeptide, H-D-Tyr(Me)-L-Ile-DL-Pip-L-Ab7-OH (1.28 g, 2.0mmol), HATU (1.14 g, 3.0 mmol), and DIEA (1.0 ml) were divided into fivealiquots. DMF (1000 ml) was added to each aliquot every 30 minutes, andcyclization reaction was carried out. After 2 hours, the reactionsolution was concentrated, dissolved in ethyl acetate, and successivelywashed with an aqueous 10% citric acid solution, an aqueous 4% sodiumbicarbonate solution, and saturated saline. After drying up over MgSO₄and concentrating, the resulting foam substance was purified by flashsilica gel chromatography (4×30 cm, 2% methanol/chloroform) to obtain afoam cyclo(-L-Ab7-D-Tyr(Me)-L-Ile-L-Pip-) (372 mg, 61%; HPLC retentiontime: 8.94 min; FAB-MS (dithiodiethanol), 607 [M+H], (607.2)) and a foamcyclo(-L-Am7(-)-D-Tyr(Me)-L-Ile-D-Pip-) (238 mg, 39%; HPLC retentiontime: 10.5 min; FAB-MS (dithiodiethanol), 607 [M+H], (607.2)).

EXAMPLE 19 Synthesis of cyclo(-L-Am7(Ac)-D-Tyr(Me)-L-Ile-L-Pip-)

Potassium thioacetate (69 mg, 0.315 mmol) was added to DMF (1 ml)containing cyclo(-L-Ab7-D-Tyr(Me)-L-Ile-L-Pip-) (130 mg, 0.21 mmol) andreacted for 3 hours. The reaction solution was concentrated byevaporation, dissolved in ethyl acetate, and then successively washedwith aqueous 10% citric acid solution and saturated saline. Thethioester produced similarly after the cyclization reaction was isolatedand purified to obtain 109 mg (86%) of oily product. HPLC retentiontime: 8.94 min; FAB-MS (dithiodiethanol), 603 [M+H], (603.3).

EXAMPLE 20 Synthesis of cyclo(-L-Am7(-)-D-Tyr(Me)-L-Ile-L-Pip-(SS-dimer:SCOP 298)

Methanol solution (0.5 ml) containingcyclo(-L-Am7(Ac)-D-Tyr(Me)-L-Ile-L-Pip-) (114 mg, 0.198 mmol) wasreacted with methanolic ammonia (10 eq.) to remove the acetyl groups.After the solvent was removed by evaporation, the residue was dissolvedin DMF (2 ml), and 1 M I₂ (ethanol) (0.25 ml) was added to the solutionfor oxidization. The produced SS-dimer was purified through a SephadexLH-20 (DMF) column and then mixed with water to obtain a white powder.The yield was 82 mg (78%). HPLC retention time: 11.6 min; HRMS (FAB,dithiodiethanol), 1063.5391 [M+H], C₅₄H₇₉O₁₀N₈S₂ (1063.5361).

EXAMPLE 21 Synthesis of cyclo(-L-Am7(Ac)-D-Tyr(Me)-L-Ile-D-Pip-)

Potassium thioacetate (69 mg, 0.60 mmol) was added to DMF (0.5 ml)containing cyclo(-L-Ab7-D-Tyr(Me)-L-Ile-D-Pip-) (240 mg, 0.40 mmol), andthis was reacted for 3 hours. The reaction solution was concentrated,dissolved in ethyl acetate, and successively washed with aqueous 10%citric acid solution, an aqueous 4% sodium bicarbonate solution, andsaturated saline. After drying over MgSO₄ and concentrating, theresulting thioester was isolated and purified in the same manner asafter the cyclization reaction to obtain an oily substance (160 mg)(66%). HPLC retention time: 10.5 min; FAB-MS (dithiodiethanol), 603[M+H], (603.3).

EXAMPLE 22 Synthesis of cyclo(-L-Am7(-)-D-Tyr(Me)-L-Ile-D-Pip-(SS-dimer:SCOP 300)

DMF (10 ml) containing cyclo(-L-Am7(Ac)-D-Tyr(Me)-L-Ile-D-Pip-) (160 mg,0.27 mmol) was reacted with methanolic ammonia (10 eq.) to remove theacetyl groups. After the solvent was removed by evaporation, the residuewas dissolved in DMF (2 ml), and 1 M I₂ (ethanol) (0.31 ml) was added tothe solution for oxidization. The produced SS-dimer was purified througha Sephadex LH-20 (DMF) column and then mixed with water to obtain awhite powder. The yield was 54 mg (36%). HPLC retention time: 13.4 min;HRMS (FAB, dithiodiethanol), 1119.5939 [M+H], C₅₈H₈₇O₁₀N₈S₂ (1119.5986).

EXAMPLE 23 Synthesis of Boc-L-Ile-D-Pro-OBzl

While cooling on ice, Boc-D-Pro-OH (17.2 g, 80 mmol) and benzyl bromide(14.3 ml, 120 mmol) were reacted in DMF (160 ml) in the presence oftriethylamine (16.8 ml, 120 mmol) to obtain Boc-D-Pro-OBzl as an oilyproduct. This product was reacted with 2 N HCl/dioxane (5 eq.) for 3hours to obtain H-D-Pro-OBzl-HCl.

While cooling on ice, DCC (8.3 g, 30 mmol) and triethylamine (3.5 ml, 25mmol) were added to DMF (200 ml) containing Boc-L-Ile-OH.1/2 H₂O (24.0g, 100 mmol), H-D-Pro-OBzl-HCl (19.3 g, 80 mmol), and HOBt.H₂O (15.3 g,100 mmol). After stirring for 8 hours, DMF was removed by evaporation,the residue was dissolved in ethyl acetate, and then successively washedwith an aqueous 10% citric acid solution, an aqueous 4% sodiumbicarbonate solution, and saturated saline. After drying over MgSO₄ andconcentrating, the resulting oily substance was purified by flash silicagel chromatography (4×30 cm, 1% methanol/chloroform) to obtain the oilytitle compound (21.5 g, 51 mmol, 72% yield). TLC: Rf=(CHCl₃/MeOH=9/1)

EXAMPLE 24 Synthesis of Boc-D-Tyr(Me)-L-Ile-D-Pro-OBzl

Boc-L-Ile-D-Pro-OBzl (21.5 g, 51.4 mmol) was dissolved in TFA (50 ml)and left with standing on ice for one hour. On completion of thereaction, TFA was removed by evaporation, and the residue wasvacuum-dried to obtain H-L-Ile-D-Pro-OBzl-TFA. The compound wasdissolved in DMF (100 ml), and Boc-D-Tyr(Me)-OH (16.7 g, 56.5 mmol) wasthen added. HBTU (29.4 g, 77 mmol), HOBt.H₂O (7.87 g, 51 mmol), andtriethylamine (25.2 ml, 180 mmol) were further added and stirred for 3hours under ice-cooling. The reaction solution was concentrated,dissolved in ethyl acetate, and successively washed with an aqueous 10%citric acid solution, an aqueous 4% sodium bicarbonate solution, andsaturated saline. The product was dried over MgSO₄ and concentrated, andthe resulting foam substance was purified by flash silica gelchromatography (4×30 cm, 1% methanol/chloroform) to obtain the foamtitle compound (22.0 g, 37 mmol, 72% yield). TLC: Rf=(CHCl₃/MeOH=9/1).

EXAMPLE 25 Synthesis of Boc-L-Ab6-OTmse

Boc-L-Ab6-OH (620 mg, 2.0 mmol) and trimethylsilylethanol (0.572 ml, 4.0mmol) were stirred in DCM (6 ml) for 6 hours in the presence of4-dimethylamino-pyridine (24.4 mg, 0.2 mmol). The reaction solution wasconcentrated, dissolved in ethyl acetate, and successively washed withaqueous 10% citric acid solution, aqueous 4% sodium bicarbonatesolution, and saturated saline. The product was dried over MgSO₄ andconcentrated, and the resulting oily substance was purified by flashsilica gel chromatography (4×30 cm, 10% ethyl acetate/hexane) to obtainan oily title compound (820 mg, 1.62 mmol, 81% yield). TLC: Rf=0.97(CHCl₃/MeOH=9/1).

EXAMPLE 26 Synthesis of Boc-D-Tyr(Me)-L-Ile-D-Pro-L-Ab6-OTmse

Boc-D-Tyr(Me)-L-Ile-D-Pro-OBzl (1.01 g, 1.70 mmol) was dissolved inmethanol (20 ml) and subjected to catalytic hydrogenation in thepresence of 5% Pd-C (150 mg). After 8 hours, the catalyst Pd-C wasfiltered and the reaction solution was evaporated to obtainBoc-D-Tyr(Me)-L-Ile-D-Pro-OH.

Boc-L-Ab6-OTmse (1.51 g, 3.0 mmol) was dissolved in TFA (5 ml) and leftstanding on ice for 30 minutes. On completion of the reaction, thereaction solution was evaporated and the residue was vacuum-dried toobtain H-L-Am6-OTmse-TFA. This product was dissolved in DMF (3.5 ml).Under ice-cooling, Boc-D-Tyr(Me)-L-Ile-D-Pro-OH (819 mg, 1.62 mmol),HATU (776 mg, 2.0 mmol), and triethylamine (0.24 ml, 1.7 mmol) weredivided into four aliquots and added to the above-described DMFsolution, which was then stirred for 3 hours. The reaction solution wasconcentrated, dissolved in ethyl acetate, and successively washed withaqueous 10% citric acid solution, aqueous 4% sodium bicarbonatesolution, and saturated saline. The resulting product was dried overanhydrous MgSO₄ and concentrated to obtain a foam substance, which wasthen purified by flash silica gel chromatography (4×30 cm, 1%methanol/chloroform) to obtain the title compound (888 mg, 1.09 mmol,64% yield). TLC: Rf=(CHCl₃/MeOH=9/1).

EXAMPLE 27 Synthesis of Boc-L-Ab7-D-Tyr(Me)-L-Ile-D-Pro-OBzl

Boc-D-Tyr(Me)-L-Ile-D-Pro-OBzl (1.19 g, 2.0 mmol) was dissolved in TFA(5 ml) and left with standing on ice for 30 minutes. On completion ofthe reaction, TFA was removed by evaporation, and the residue wasvacuum-dried to obtain H-D-Tyr(Me)-L-Ile-D-Pro-OBzl-TFA. The compoundwas dissolved in DMF (4.0 ml), and Boc-L-Ab7-OH (652 mg, 2.0 mmol) wasthen added. HBTU (1.14 g, 3.0 mmol), HOBt.H₂O (306 mg, 2.0 mmol), andtriethylamine (1.4 ml, 10 mmol) were further added and stirred for 3hours under ice-cooling. The reaction solution was concentrated,dissolved in ethyl acetate, and successively washed with an aqueous 10%citric acid solution, an aqueous 4% sodium bicarbonate solution, andsaturated saline. The product was dried over MgSO₄ and concentrated, andthe resulting foam substance was purified by flash silica gelchromatography (4×30 cm, 2% methanol/chloroform) to obtain the foamtitle compound (1.51 g, 1.89 mmol, 94% yield). HPLC retention time: 9.15min.

EXAMPLE 28 Synthesis of Boc-L-Ab8-D-Tyr(Me)-L-Ile-D-Pro-OBzl

Boc-D-Tyr(Me)-L-Ile-D-Pro-OBzl (1.19 g, 2.0 mmol) was dissolved in TFA(5 ml) and left with standing on ice for 30 minutes. On completion ofthe reaction, TFA was removed by evaporation, and the residue wasvacuum-dried to obtain H-D-Tyr(Me)-L-Ile-D-Pro-OBzl.TFA. The compoundwas dissolved in DMF (4.0 ml), and Boc-L-Ab8-OH (676 mg, 2.0 mmol) wasthen added. HBTU (1.14 g, 3.0 mmol), HOBt.H₂O (306 mg, 2.0 mmol), andtriethylamine (.1.4 ml, 10 mmol) were further added and stirred for 3hours under ice-cooling. The reaction solution was concentrated,dissolved in ethyl acetate, and successively washed with an aqueous 10%citric acid solution, an aqueous 4% sodium bicarbonate solution, andsaturated saline. The product was dried over MgSO₄ and concentrated, andthe resulting foam substance was purified by flash silica gelchromatography (4×30 cm, 2% methanol/chloroform) to obtain the foamtitle compound (1.44 g, 1.76 mmol, 88% yield). HPLC retention time: 10.9min.

EXAMPLE 29 Synthesis of Boc-L-Ab9-D-Tyr(Me)-L-Ile-D-Pro-OBzl

Boc-D-Tyr(Me)-L-Ile-D-Pro-OBzl (1.19 g, 2.0 mmol) was dissolved in TFA(5 ml) and left with standing on ice for 30 minutes. On completion ofthe reaction, TFA was removed by evaporation, and the residue wasvacuum-dried to obtain H-D-Tyr(Me)-L-Ile-D-Pro-OBzl.TFA. The compoundwas dissolved in DMF (4.0 ml), and Boc-L-Ab9-OH (775 mg, 2.2 mmol) wasthen added. HBTU (1.14 g, 3.0 mmol), HOBt.H₂O (306 mg, 2.0 mmol), andtriethylamine (1.4 ml, 10 mmol) were further added and stirred for 3hours under ice-cooling. The reaction solution was concentrated,dissolved in ethyl acetate, and successively washed with an aqueous 10%citric acid solution, an aqueous 4% sodium bicarbonate solution, andsaturated saline. The product was dried over MgSO₄ and concentrated, andthe resulting foam substance was purified by flash silica gelchromatography (4×30 cm, 2% methanol/chloroform) to obtain the foamtitle compound (1.31 g, 1.58 mmol, 79% yield). HPLC retention time: 11.7min.

EXAMPLE 30 Synthesis of H-D-Tyr(Me)-L-Ile-D-Pro-L-Ab6-OH.TFA

Boc-D-Tyr(Me)-L-Ile-D-Pro-L-Ab6-OTmse (888 mg, 1.11 mmol) was dissolvedin ethanol (10 ml). Under ice-cooling, an aqueous 1 N NaOH solution(1.32 ml, 1.33 mmol) divided into three aliquots was added to thesolution and left standing on ice for 3 hours. The reaction solution wasconcentrated, dissolved in ethyl acetate, and successively washed with10% citric acid and saturated saline. After drying over MgSO₄, theproduct was concentrated to obtain Boc-D-Tyr(Me)-L-Ile-D-Pro-L-Ab6-OH.The compound was dissolved in TFA (5 ml) and left standing on ice for 30minutes. The reaction solution was evaporated, and the residue wasvacuum-dried to obtain an oily title compound (778 mg, 1.07 mmol, 96%yield).

EXAMPLE 31 Synthesis of H-L-Ab7-D-Tyr(Me)-L-Ile-D-Pro-OH.TFA

Boc-L-Ab7-D-Tyr(Me)-L-Ile-D-Pro-OBzl (1.51 g, 1.89 mmol) was dissolvedin methanol (5 ml) and subjected to catalytic hydrogenation in thepresence of 5% Pd-C (150 mg). After 5 hours, the catalyst Pd-C wasfiltered and the reaction solution was evaporated to obtainBoc-L-Ab7-D-Tyr(Me)-L-Ile-D-Pro-OH. The resulting compound was dissolvedin TFA (5 ml) and left standing on ice for 30 minutes. After thereaction solution was evaporated, the residue was vacuum-dried to obtainthe oily title compound (1.15 mg, 1.84 mmol, 97% yield).

EXAMPLE 32 Synthesis of H-L-Ab8-D-Tyr(Me)-L-Ile-D-Pro-OH.TFA

Boc-L-Ab8-D-Tyr(Me)-L-Ile-D-Pro-OBzl (1.44 g, 1.76 mmol) was dissolvedin methanol (5 ml) and subjected to catalytic reduction in the presenceof 5% Pd-C (150 mg). After 5 hours, the catalyst Pd-C was filtered andthe reaction solution was evaporated to obtainBoc-L-Ab8-D-Tyr(Me)-L-Ile-D-Pro-OH. The resulting compound was dissolvedin TFA (5 ml) and left standing on ice for 30 minutes. After thereaction solution was evaporated, the residue was vacuum-dried to obtainthe oily title compound (1.15 mg, 1.84 mmol, 97% yield).

EXAMPLE 33 Synthesis of H-L-Ab9-D-Tyr(Me)-L-Ile-D-Pro-OH.TFA

Boc-L-Ab9-D-Tyr(Me)-L-Ile-D-Pro-OBzl (1.31 g, 1.58 mmol) was dissolvedin methanol (2 ml) and subjected to catalytic hydrogenation in thepresence of 5% Pd-C (150 mg). After 12 hours, the catalyst Pd-C wasfiltered and the reaction solution was evaporated to obtainBoc-L-Ab9-D-Tyr(Me)-L-Ile-D-Pro-OH. This compound was dissolved in TFA(5 ml) and left standing on ice for 30 minutes. After the reactionsolution was evaporated, ether/petroleum ether (1:10) was added to theresidue for solidification. This was then vacuum-dried to obtain thetitle compound (905 mg, 1.42 mmol, 90% yield).

EXAMPLE 34 Synthesis of cyclo(-L-Ab6-D-Tyr(Me)-L-Ile-D-Pro-)

H-D-Tyr(Me)-L-Ile-D-Pro-L-Ab6-OH.TFA (778 mg, 1.07 mmol), HATU (616 mg,1.62 mmol), and DIEA (0.75 ml) were divided into five aliquots and addedto DMF (110 ml) every 30 minutes to carry out a cyclization reaction.After 2 hours, the solvent was removed by evaporation. The residue wasdissolved in ethyl acetate, successively washed with 10% citric acid, 4%NaHCO₃, and saline. The product was dried over MgSO₄ and concentrated,and the resulting foam substance was purified by flash silica gelchromatography (4×30 cm, 1% methanol/chloroform) to obtain a colorlessoily compound (146 mg) (23%). HPLC retention time: 9.06 min; HRMS (FAB,dithiodiethanol), 579.2197 [M+H], C₂₇H₄₁O₅N₄ ⁷⁹Br (579.2182).

This time, a cyclic tetrapeptide containing an HOAt adduct transferredby substituting the Ab6 side chain terminus Br,cyclo(-L-A(OAt)6-D-Tyr(Me)-L-Ile-D-Pro-) (167 mg) (27%), was obtained asa foam. HPLC retention time: 8.16 min; HRMS (FAB, dithiodiethanol),635.3312 [M+H], C₃₂H₄₃O₆N₈ (635.3306)

EXAMPLE 35 Synthesis of cyclo(-L-Ab7-D-Tyr(Me)-L-Ile-D-Pro-)

H-L-Ab7-D-Tyr(Me)-L-Ile-D-Pro-OH.TFA (1.15 g, 1.84 mmol), HATU (1.05 g,2.76 mmol), and DIEA (1.28 ml) were divided into five aliquots and addedto DMF (180 ml) every 30 minutes to carry out a cyclization reaction.The product was purified in the same manner as described above,resulting in a foam (700 mg) (64%). HPLC retention time: 9.90 min; HRMS(FAB, dithiodiethanol), 593.2300 [M+H], C₂₈H₄₂O₅N₄ ⁷⁹Br (593.2339).

EXAMPLE 36 Synthesis of cyclo(-L-Ab8-D-Tyr(Me)-L-Ile-D-Pro-)

H-L-Ab8-D-Tyr(Me)-L-Ile-D-Pro-OH.TFA (512 mg, 0.80 mmol), HATU (455 mg,1.20 mmol), and DIEA (0.56 ml) were divided into five aliquots and addedto DMF (80 ml) every 30 minutes to carry out a cyclization reaction.After 2 hours, the reaction solution was concentrated, dissolved inethyl acetate, and successively washed with aqueous 10% citric acidsolution, aqueous 4% sodium bicarbonate solution, and saturated saline.This was then dried over MgSO₄ and concentrated, and the resulting foamsubstance was purified by flash silica gel chromatography (4×30 cm, 1%methanol/chloroform) to obtain a foam (267 mg) (55%). HPLC retentiontime: 9.95 min; HRMS (FAB, dithiodiethanol), 607.2501 [M+H], C₂₉H₄₄O₅N₄⁷⁹Br (607.2495).

EXAMPLE 37 Synthesis of cyclo(-L-Ab9-D-Tyr(Me)-L-Ile-D-Pro-)

H-L-Ab9-D-Tyr(Me)-L-Ile-D-Pro-OH.TFA (905 mg, 1.41 mmol), HATU (833 mg,2.12 mmol), and DIEA (0.64 ml) were divided into five aliquots and addedto DMF (150 ml) every 30 minutes to carry out a cyclic reaction. Aftertwo hours, the reaction solution was concentrated, dissolved in ethylacetate, and successively washed with aqueous 10% citric acid solution,aqueous 4% sodium bicarbonate solution, and saturated saline. This wasthen dried with MgSO₄ and concentrated, and the resulting foam substancewas purified by flash silica gel chromatography (4×30 cm, 1%methanol/chloroform) to obtain a foam (533 mg) (61%). HPLC retentiontime: 10.9 min; HRMS (FAB, dithiodiethanol), 621.2625 [M+H], C₃₀H₄₆O₅N₄⁷⁹Br (621.2652).

EXAMPLE 38 Synthesis of cyclo(-L-Am6(Ac)-D-Tyr(Me)-L-Ile-D-Pro-)

Potassium thioacetate (57.6 mg, 0.504 mmol) was added to DMF (0.5 ml)containing cyclo(-L-Ab6-D-Tyr(Me)-L-Ile-D-Pro-) (146 mg, 0.252 mmol),and this was reacted for 3 hours. The reaction solution wasconcentrated, dissolved in ethyl acetate, and successively washed withaqueous 10% citric acid solution and saturated saline. After drying overMgSO₄ and concentrating, the resulting thioester was isolated andpurified in the same manner as after the cyclic reaction to obtain anoily substance (114 mg) (79%). HPLC retention time: 9.06 min; HRMS (FAB,dithiodiethanol), 575.2879 [M+H], C₂₉H₄₃O₆N₄S (575.2903).

EXAMPLE 39 Synthesis of cyclo(-L-Am7(Ac)-D-Tyr(Me)-L-Ile-D-Pro-)

Potassium thioacetate (52 mg, 0.452 mmol) was added to DMF (0.5 ml)containing cyclo(-L-Ab7-D-Tyr(Me)-L-Ile-D-Pro-) (133 mg, 0.226 mmol),and this was reacted for 3 hours. The reaction solution wasconcentrated, dissolved in ethyl acetate, and successively washed withaqueous 10% citric acid solution and saturated saline. After drying overMgSO₄ and concentrating, the resulting thioester was isolated andpurified to obtain an oily substance (118 mg). (89%). HPLC retentiontime: 9.90 min; HRMS (FAB, dithiodiethanol), 589.3605 [M+H], C₃₀H₄₅O₆N₄S(589.3060).

EXAMPLE 40 Synthesis of cyclo(-L-Am8(Ac)-D-Tyr(Me)-L-Ile-D-Pro-)

Potassium thioacetate (100 mg, 0.878 mmol) was added to DMF (1 ml)containing cyclo(-L-Ab8-D-Tyr(Me)-L-Ile-D-Pro-) (267 mg, 0.439 mmol),and this was reacted for 3 hours. The reaction solution wasconcentrated, dissolved in ethyl acetate, and successively washed withaqueous 10% citric acid solution and saturated saline. After drying overMgSO₄ and concentrating, the resulting thioester was isolated andpurified to obtain an oily substance (222 mg) (84%). HPLC retentiontime: 9.95 min; HRMS (FAB, dithiodiethanol), 603.3244 [M+H], C₃₁H₄₇O₆N₄S(575.2903).

EXAMPLE 41 Synthesis of cyclo(-L-Am9(Ac)-D-Tyr(Me)-L-Ile-D-Pro-)

Potassium thioacetate (91.4 mg, 0.804 mmol) was added to DMF (0.5 ml)containing cyclo(-L-Ab9-D-Tyr(Me)-L-Ile-D-Pro-) (250 mg, 0.402 mmol),and this was reacted for 3 hours. The reaction solution wasconcentrated, dissolved in ethyl acetate, and successively washed withaqueous 10% citric acid solution and saturated saline. After drying overMgSO₄ and concentrating, the resulting thioester was isolated andpurified to obtain an oily substance (190 mg) (77%). HPLC retentiontime: 10.9 min; HRMS (FAB, dithiodiethanol), 617.3364 [M+H], C₃₂H₄₉O₆N₄S(617.3373).

EXAMPLE 42 Synthesis of cyclo(-L-Am6(-)-D-Tyr(Me)-L-Ile-D-Pro-SS-dimer(SCOP 302)

Methanol (0.5 ml) containing cyclo(-L-Am6(Ac)-D-Tyr(Me)-L-Ile-D-Pro-)(114 mg, 0.198 mmol) was reacted with methanolic ammonia (10 eq.) toremove the acetyl groups. After the solvent was removed by evaporation,the residue was dissolved in DMF (2 ml), to which 1 M I₂ (ethanol) (0.2ml) was added for oxidization. The SS-dimer produced was purifiedthrough a Sephadex LH-20 (DMF) column, and then mixed with water toobtain white powder. The yield was 82 mg (78%). HPLC retention time:11.6 min; HRMS (FAB, dithiodiethanol), 1063.5391 [M+H], C₅₄H₇₉O₁₀N₈S₂(1063.5361).

EXAMPLE 43 Synthesis of cyclo(-L-Am7(-)-D-Tyr(Me)-L-Ile-D-Pro-) SS-dimer(SCOP 304)

Methanol (0.5 ml) containing cyclo (-L-Am7(Ac)-D-Tyr(Me)-L-Ile-D-Pro-)(118 mg, 0.201 mmol) was reacted with methanolic ammonia to remove theacetyl groups of the compounds. The terminal sulfohydryl groups of thecompounds were oxidized by adding 1 M I₂ (ethanol). After purification,SS-dimer was obtained as white powder. 98 mg (89%) yield. HPLC retentiontime: 12.3 min; HRMS (FAB, dithiodiethanol), 1091.5684 [M+H],C₅₆H₈₃O₁₀N₈S₂ (1091.5674)

EXAMPLE 44 Synthesis of cyclo(-L-Am8(-)-D-Tyr(Me)-L-Ile-D-Pro-SS-dimer(SCOP 306)

Methanol (0.5 ml) containing cyclo(-L-Am8(Ac)-D-Tyr(Me)-L-Ile-D-Pro-)(222 mg, 0.368 mmol) was reacted with methanolic ammonia to remove theacetyl groups of the compounds. The terminal sulfohydryl groups of thecompounds were oxidized by adding 1 M I₂ (ethanol). After purification,SS-dimer was obtained as white powder. 167 mg (81%) yield. HPLCretention time: 13.0 min; HRMS (FAB, dithiodiethanol), 1119.5961 [M+H],C₅₆H₈₆O₁₀N₈S₂ (1119.5987)

EXAMPLE 45 Synthesis of cyclo(-L-Am9(-)-D-Tyr(Me)-L-Ile-D-Pro-SS-dimer(SCOP 308)

Methanol (0.5 ml) containing cyclo(-L-Am9(Ac)-D-Tyr(Me)-L-Ile-D-Pro-)(95 mg, 0.154 mmol) was reacted with methanolic ammonia to remove theacetyl groups of the compounds. The terminal sulfohydryl groups of thecompounds were oxidized by adding 1 M I₂ (ethanol). After purification,SS-dimer was obtained as white powder. 84 mg (98%) yield. HPLC retentiontime: 14.2 min; HRMS (FAB, dithiodiethanol), 1147.6307 [M+H],C₆₀H₉₁O₁₀N₈S₂ (1147.6300).

EXAMPLE 46 Synthesis of cyclo(-L-Am7(SMEt)-D-Tyr(Me)-L-Ile-D-Pro-) (SShybrid: SCOP 404)

DMF (0.5 ml) containing cyclo(-L-Am7(Ac)-D-Tyr(Me)-L-Ile-D-Pro-) (270mg, 0.45 mmol) was reacted with methanolic ammonia (10 eq.) to removethe acetyl groups of the compounds. After ammonia was removed byevaporation, 2-mercaptoethanol (10 eq.) and then 1 M I₂ (ethanol) 0.2 mlwere added to the residue, causing oxidation. The resulting SS-hybridwas purified by a Sephadex LH-20 (DMF) column and freeze-dried to obtainthe title compound as a white powder. The yield was 30 mg (11%). HPLCretention time: 8.9 min; HRMS (FAB, dithiodiethanol), 622.2877 [M],C₃₀H₄₆O₆N₄S₂ (622.2859).

EXAMPLE 47 Synthesis of cyclo(-L-Am7(S2Py)-D-Tyr(Me)-L-Ile-D-Pro-) (SShybrid: SCOP 401)

DMF (1 ml) containing cyclo(-L-Am7(Ac)-D-Tyr(Me)-L-Ile-D-Pro-) (40 mg,0.07 mmol) was mixed with 2,2′-dithiopyridine (31 mg, 0.14 mmol) andmethanolic ammonia (10 eq.) and stirred for 8 hours. After the reactionsolution was concentrated, the powder product was purified by flashsilica gel chromatography (4×30 cm, 1% methanol/chloroform) to obtainthe title compound. The yield was 15 mg (38%). HPLC retention time: 9.6min; HRMS (FAB, dithiodiethanol), 656.2952 [M+H], C₃₃H₄₅O₅N₅S₂(656.2940).

EXAMPLE 48 Synthesis of cyclo(-L-Am7(S4Py)-D-Tyr(Me)-L-Ile-D-Pro-) (SShybrid: SCOP 402)

DMF (1 ml) containing cyclo(-L-Am7(Ac)-D-Tyr(Me)-L-Ile-D-Pro-) (100 mg,0.17 mmol) was mixed with 4,4′-dithiopyridine (75 mg, 0.34 mmol) andmethanolic ammonia (20 eq.) and stirred for 8 hours. After the reactionsolution was concentrated, the powder product was purified by flashsilica gel chromatography (4×30 cm, 1% methanol/chloroform) to obtainthe title compound. The yield was 13 mg (13%). HPLC retention time: 6.5min; HRMS (FAB, dithiodiethanol), 656.2934 [M+H], C₃₃H₄₅O₅N₅S₂(656.2940).

EXAMPLE 49 Synthesis of cyclo(-L-Am7(SEll)-D-Tyr(Me)-L-Ile-D-Pro-) (SShybrid: SCOP 403)

DMF (2.8 mL) containing 5,5′-dithiobis(2-nitrobenzoic acid) (515 mg, 1.4mmol) was mixed with dimethylamine (343 mg, 3.0 mmol), DCC (867 mg, 3.0mmol), and HOBt.H₂O (214 mg, 1.4 mmol), and then stirred for eight hourswhile cooling on ice. On completion of the reaction, the reactionsolution was concentrated, dissolved in ethyl acetate, and successivelywashed with an aqueous 10% citric acid solution, an aqueous 4% sodiumbicarbonate solution, and saturated saline. After drying over MgSO₄,ethyl acetate was removed by evaporation. The residue was vacuum-driedand purified using flash silica gel chromatography (4×30 cm, 1%methanol/chloroform) to obtain 5,5′-dithiobis(2-nitrobenzoic aciddimethylamide).

DMF (2 ml) containing cyclo(-L-Am7(Ac)-D-Tyr(Me)-L-Ile-D-Pro-) (130 mg,0.22 mmol) was mixed with 5,5′-dithiobis(2-nitrobenzoic aciddimethylamide) (198 mg, 0.44 mmol) and methanolic ammonia (10 eq.), andthen stirred for 6 hours. The reaction solution was concentrated,dissolved in a small amount of DMF, and purified by HPLC (column:YMC-Pack ODS-A 10×250 mm) to obtain the title compound. The yield was 13mg (9.3%). HPLC retention time: 9.5 min; HRMS (FAB, dithiodiethanol),771.3201 [M+H], C₃₇H₅₀O₈N₆S₂ (771.3210).

EXAMPLE 50 Synthesis of cyclo(-L-Am7(SMe)-D-Tyr(Me)-L-Ile-D-Pro-) (SCOP405)

DMF (1 ml) containing cyclo(-L-Ab7-D-Tyr(Me)-L-Ile-D-Pro-) (118 mg, 0.2mmol) was mixed with 4-methoxybenzylmercaptan (0.056 ml, 0.4 mmol) andtriethylamine (0.07 ml, 0.5 mmol) and reacted at a room temperature for2 hours. The produced cyclo(-L-Am7(Mb)-D-Tyr(Me)-L-Ile-D-Pro-) wasextracted with ethyl acetate, purified, and then reacted withdimethyl(methylthio)sulfonium tetrafluoroborate (0.9 mmol, 176 mg) inmethanol (18 ml) at room temperature for 2 hours. The reaction solutionwas concentrated, dissolved in chloroform, and purified by silica gelchromatography (2×25 cm, 2% methanol/chloroform) to obtain the targetproduct. The yield was 69 mg (65%). TLC Rf: 0.90 (CHCl₃/MeOH=19/1). HPLCretention time: 12.28 min; HR-FAB+MS: 593.2777 (calcd.: 592.2753,composition: C₂₉H₄₄O₅N₄S₂, matrix: 2,2′-ditihidiethanol).

EXAMPLE 51 Measurement of HDAC Inhibition Activity

In this Example, the HDAC inhibition activity of SCOP was measured. FIG.1 to FIG. 4 show lists of the structures of the sulfur-containing cyclicpeptides (SCOP) whose activity was measured. The conformation and numberof carbon chains until the active groups of the cyclic tetrapeptidestructures were investigated based on natural HDAC inhibitors, Cyl-1 andCyl-2, as shown in FIG. 5 (Furumai et al. (2001) Proc. Natl. Acad. Sci.USA, 98, 87-92).

The steric conformation of natural Cyl-1 and Cyl-2 is LDLL, howeverthose with LDLD conformation were also investigated. In the followingexperimental results, DTT is coexisted for X=H, for the purpose ofcutting disulfide bonds.

To measure HDAC inhibition activity, an HDAC solution was prepared asdescribed below. 1×10⁷ of 293T cells were plated on to a 100-mm dishand, after 24 hours, transfected with vectors (1 μg) expressing humanHDAC1 and HDAC4 or mouse HDAC6, using LipofectAmine 2000 reagent (LifeTechnologies, Inc. Gaithersburg, Md.). The above-mentioned pcDNA3-HD1was used as a vector expressing human HDAC1 (Yang, W. M., Yao, Y. L.,Sun, J. M., Davie, J. R. & Seto, E. (1997) J. Biol. Chem. 272,28001-28007). pcDNA3.1(+)-HD4 was used as a vector expressing humanHDAC4 (Fischle, W., Emiliani, S., Hendzel, M. J., Nagase, T., Nomura,N., Voelter, W. & Verdin, E. (1999) J. Biol. Chem. 274, 11713-11720).pcDNA-mHDA2/HDAC6 was used as a vector expressing mouse HDAC6 (Verdel,A. & Khochbin, S. (1999) J. Biol. Chem. 274, 2440-2445). The vectorswere introduced for five hours in OPTI-MEM. The medium was then replacedwith Dulbecco's modified Eagle's medium (DMEM), and incubated for 19hours. The cells were washed with PBS, suspended in lysis buffer (50 mMTris-HCl (pH7.5), 120 mM NaCl, 5 mM EDTA, and 0.5% Nonidet P-40), andsonicated. The supernatant was collected by centrifugation andnonspecific protein was removed using Protein A/G plus agarose beads(Santa Cruz Biotechnologies, Inc.). Anti-FLAG M2 antibodies(Sigma-Aldrich, Inc.) were added to the supernatant of cells expressingHDAC1 or HDAC4. Anti-HA antibodies (clone 3F10, Roche MolecularBiochemicals) were added to the supernatant of cells expressing HDAC6.Reaction in the respective mixtures was carried out at 4° C. for onehour. The resulting reaction mixtures were independently mixed withagarose beads and further reacted at 4° C. for one hour. The agarosebeads were washed three times with lysis buffer and then washed oncewith HD buffer (20 mM Tris-HCl (pH8.0), 150 mM NaCl, 10% glycerol, and acomplete protease inhibitor cocktail (Boehringer Mannheim, Germany)).The protein solution, referred to as “HDAC reaction solution”, that hadbonded to the agarose beads was recovered by incubation with FLAGpeptide (40 μg) (Sigma-Aldrich, Inc.) or HA peptide (100 μg) in an HDbuffer (200 μl) at 4° C. for one hour. The HDAC reaction solution wasused for determining HDAC inhibition activity as shown below.

In vitro HDAC inhibition activity was evaluated as follows: A testcompound was dissolved in DMSO and adjusted to 10 mM. This was used asan inhibitor stock solution. As a positive control, Tricostatin A (TSA),known as an HDAC inhibitor, was dissolved in DMSO to obtain a 10 mMstock solution. Measurement was carried out by incubating each of theabove-mentioned HDAC solutions and a solution of acetylated histonesubstrate labeled with [³H] at 37° C. for 15 minutes (100 μl reactionvolume) in the presence of a test compound or control TSA. Thesereactions were stopped by adding 10 μl HCl. The [³H] acetic acid excisedby the enzyme reaction was extracted with ethyl acetate and subjected toradioactive dose measurement. As a negative control, the same procedurewas carried out in which no inhibitor was added to the reaction system.The inhibition activity was expressed as a 50% inhibition concentration(“IC50 (nM)”) of the HDAC activity in the negative control (Tables 1 to4).

The HDAC inhibition activity in vivo was measured using p21promoter-inducing activity as an index, as shown below. The MFLL-9 cellsemployed for the experiments stably maintained fusion genes of humanwild-type p21 promoter and luciferase (Dr. B. Vogelstaein). Using phenolred-free DMEM medium comprising 10% FBS, cultivation was carried out ina steam-saturated incubator at 37° C. with 5% carbon dioxide. The MFLL-9cells were plated at a density of 85,000 cells/well on a 96-wellmicrotiter plate, each in 99 μl of the above-mentioned medium. Thesewere then cultivated for six hours. One μl of test compound solution wasadded to each well, which was then cultured for another 18 hours. TSAwas used as the positive control compound with p21 promoter-inducingactivity, which results from HDAC inhibition activity.

The intensity of luminescence caused by the product of the enzymereaction for intracellular luciferase expression was measured using LucLite (Packard BioScience Company). A group in which test compounds werenot added was used as a negative control group. The values measured forthis group were used as a standard. The activities for eachconcentration of added test compound were expressed relative to theabove-mentioned standard value, 1. The test compound activityintensities were compared using the concentrations (“EC50 (nM)”)corresponding to 50% of the maximum active values for TSA (Tables 1 to4). TABLE 1 X = H (coexisting with DTT) Inhibitor IC50 (nM) P21 PromoterNumber of SCOP No. HDAC1 HDAC4 HDAC6 EC50 (nM) Conformation CarbonChains 148 81.4 17.0 >500000 6720 Cyl1 (LDLL) C5 149 2.37 5.22 44300 596Cyl2 (LDLL) C5 150 2.10 4.26 5560 504 Cyl2 (LDLD) C5 151 932 734028500 >100000 Cyl1 (LDLD) C4 152 4.60 2.06 1400 309 Cyl1 (LDLD) C5 1539.13 91.0 8050 9850 Cyl1 (LDLD) C6 154 38.1 99.2 2470 31400 Cyl1 (LDLD)C7

As the in vitro inhibition activity and in vivo P21 promoter activity ofTable 1 shows, LDLD isomers were found to have higher activities thanLDLL isomers which are in a natural conformation. In addition, C5 wasshown to be most preferable as the number of carbon chains until theactive thiol group. TABLE 2 X = a compound comprising the leftconformation (homodimer) Number of Inhibitor IC50 (nM) P21 PromoterCarbon SCOP No. HDAC1 HDAC4 HDAC6 EC50 (nM) Conformation Chains 296 763222 >500000 7730 Cyl1 (LDLL) C5 298 114 33.7 418000 5800 Cyl2 (LDLL) C5300 61.1 36.2 255000 7370 Cyl2 (LDLD) C5 3027200 >500000 >500000 >100000 Cyl1 (LDLD) C4 304 142 145 >500000 341 Cyl1(LDLD) C5 306 153 319 1320000 847100000 Cyl1 (LDLD) C6 308 983 505745000 235000 Cyl1 (LDLD) C7

As for the case of X=H, with respect to the homodimers, it was shownthat LDLD isomers have higher activities than LDLL isomers which are ina natural conformation, and that C5 is the most preferable number ofcarbon chains until the active thiol group. TABLE 3 X = a lowmolecular-weight compound (hybrid) P21 Promoter Inhibitor IC50 (nM) EC50SCOP No. HDAC1 HDAC4 HDAC6 (nM) Conformation 401 NT NT NT 1360 152 +2-Pyridine 402 6.76 68.3 1610 1310 152 + 4-Pyridine 403 21.5 18.9 60801800 152 + Ellman's reagent 404 217 355 201000 1360 152 +Mercaptoethanol 405 119 405 191 3260 152 + Methylmercaptane 401/DTT NTNT NT 815 402/DTT 0.553 1.12 2010 470 403/DTT 1.15 1.53 4730 748 404/DTT2.44 13.0 15400 754“NT” means that no test was carried out.

Even hybrid bodies of SCOP 152 and low molecular-weight compounds wereshown to have inhibition activity. TABLE 4 Positive control (TSA) P21Inhibitor IC50 (nM) Promoter TSA HDAC1 HDAC4 HDAC6 EC50 (nM) TSA 19.268.3 27.2 445

According to the above results, LDLD isomers have higher activities thanLDLL isomers in their natural conformation. In addition, C5 was found tobe the most preferable number of carbon chains until the active thiolgroup. Furthermore, since the HDAC6 inhibition activity wassignificantly low, the compounds were confirmed to comprise enzymesubtype-selective inhibition activity. On an enzymatic level, thepresent compounds showed high HDAC inhibition activity when they werethiols (X=H) coexisting with DTT. However, on a cellular level, eventhose X=left structure or X=low molecular-weight compounds showed highactivity. This suggests that thiol groups were exposed and thusactivated by reducing the disulfides incorporated into cells usingintracellular reducing forces.

Next, since it was possible that DTT had some effect, an experiment wascarried out using purified SCOP 152, under DTT-free conditions. TABLE 5P21 IC50 (nM) Promoter Inhibitor HDAC1 HDAC4 HDAC6 EC50 (nM) 152 NT NTNT 3510“NT” means that no test was carried out.

The EC50 value increased compared to when DTT was present. DTT'sexistence was thought to reduce the pH of the culture medium, causing achange in monomer stability. Alternatively, it may be also possible thatDTT served as a protection group. The following experiment was carriedout using purified SCOP 152 under the DTT-free conditions.

EXAMPLE 52 Measurement of in vivo HDAC Inhibition Activity

Histone acetylation levels were measured by: (i) reacting a testcompound with HeLa cells; and (ii) confirming the histone acetylationlevel by Western blotting using anti-acetylated lysine antibodies.Specifically, human uterine cancer cells (HeLa) were cultured in a DMEMmedium comprising 10% FBS at 37° C. in the presence of 5% carbon dioxidein a steam-saturated incubator. Two ml of the cells at a density of15,000 cells/ml were plated onto a 6-well plate and cultured for 18hours. Test compound solution was added to each culture and successivelycultured for another six hours. The cells were washed with PBS,suspended in a lysis buffer (50 mM Tris-HCl (pH7.5), 120 mM NaCl, 5 mMEDTA, 0.5% Nonidet P-40), and then sonicated. The supernatant wascollected by centrifugation, mixed with SDS buffer, and left at 100° C.for five minutes. The resulting sample was subjected to electrophoresison a 15% SDS gel and transferred to a membrane film. This was treatedwith primary antibody “AKL5C1” (Japan Energy), and secondary antibody“anti-mouse” (LIFE SCIENCE), and then acetylation bands were detected byECL (amersham pharmacia biotech) (FIG. 6). The concentration unit of thecompounds shown in FIG. 6 is “nM”.

As shown in FIG. 6, the inhibition tendencies shown were the same as theresults (EC50) of P21 promoter-inducing activity. C5 was the mostpreferable number of carbon chains until the active thiol group.

EXAMPLE 53 Cytotoxicity Test

Human normal lung cells (TIG-3) and human uterine cancer cells (HeLa)were used to test SCOP cytotoxicity. These TIG-3 and HeLa cells werecultured in a DMEM medium comprising 10% FBS at 37° C. in the presenceof 5% carbon dioxide in a steam-saturated incubator. The TIG-3 and HeLacells were plated in 100 μl/well of the above-described medium on a96-well microtiter plate, at a density of 30,000 cells/well and 10,000cells/well respectively. This was then cultured for 18 hours. Testcompound solution diluted with medium was added to each well and culturewas continued for another 48 hours.

30 μl of supernatant from each well was transferred to another 96-wellmicrotiter plate (A), and the remaining supernatant was discarded. 100μl of 0.5% Triton-X/PBS was added to each well to lyse the cells, and 30μl was then transferred to each respective well of another 96-wellmicrotiter plate (B). 30 μl of LDH-Cytotoxic Test (Wako) substratesolution was added to each well of these 96-well microtiter plates A andB, causing a color reaction. Once the color reaction was sufficientlyprogressed, it was stopped by adding 60 μl of a quenching solution.Color intensity was measured at OD560 nm using a microplate reader(Softmax). [A/(A+B)] was calculated as the free-LDH ratio. Inhibitionactivity was shown as LD50 when the free-LDH ratio was 50%. The higherthe activity value for cancer-cell-selective cell damage (LD50 fornormal cells/LD50 for cancer cells), the more that cancer-cell-selectiveapoptosis was induced. TABLE 6 LD50 (nM) Cancer-Cell-Selective InhibitorHeLa TIG-3 Cytotoxicity TSA 41.4 1580 38.2 SCOP 152 370 6780 18.3 SCOP304 151 3471 23.0 SCOP 402 1170 13300 11.4 SCOP 405 179 7900 44.1 SCOP304/DTT 47.1 1190 25.2 SCOP 402/DTT 161 4460 27.8

As shown in Table 6, the compounds of the present invention wereconfirmed to have intense cancer-cell-selective cytotoxicity that is aseffective as TSA.

EXAMPLE 54 Evaluation of Stability

The stability of SCOP 152, SCOP 304, and SCOP 402 in serum was evaluatedby the method shown below: 1 μl of 10 mM SCOP 152, SCOP 304, and SCOP402 was added to 99 μl FCS, and incubated at 37° C. Each hour, NaCl in asufficient amount for saturation and 1 ml of ethyl acetate were added toeach mixture. After extraction, from 800 μl of the ethyl acetate phase,the ethyl acetate was distilled off, and then 100 μl of DMSO was addedto the residue. The resulting solution was further diluted ten timeswith DMSO and used to measure p21 promoter inducing activity. Activityat incubation time zero was taken as 100%, and activities were compared(FIG. 7).

As shown in FIG. 7, SCOP 304 and SCOP 402 were able to stably retainactivity in serum for longer than SCOP 152. This stability was thoughtto be improved by the protection of thiol groups.

Next, in vivo stability was investigated based on histone acetylationlevels. HeLa cells were treated with each compound and then histoneacetylation level was analyzed by Western blotting using ananti-acetylated lysine antibody (FIG. 8). Specifically, human uterinecancer cells (HeLa) were cultured in DMEM medium comprising 10% FBS at37° C. with 5% carbon dioxide in a steam-saturated incubator. Two ml ofthe cells were plated at a density of 15,000 cells/ml in a 6-well plate,and cultured for 18 hours. 200 nM of test compound solutions comprisingTSA, SCOP 152 and SCOP 304, and 1 μM test compound SCOP 402 solutionwere added, and culture was continued for an appropriate time. The cellswere washed with PBS, suspended in a lysis buffer (50 mM Tris-HCl(pH7.5), 120. mM NaCl, 5 mM EDTA, and 0.5% Nonidet P-40), and thensonicated. Each supernatant was collected by centrifugation, mixed witha SDS buffer, and treated at 100° C. for five minutes. The resultingsample was subjected to electrophoresis on a 15% SDS gel and transferredto a membrane film. After treatment with primary antibody “AKL5C1”(Japan Energy), and secondary antibody “anti-mouse” (LIFE SCIENCE), ECL(amersham pharmacia biotech) treatment was carried out and acetylationbands were detected.

The compounds of the present invention showed intense inhibitionactivity towards HDAC1 and HDAC4, but scarcely any inhibition activitytowards HDAC6. HDAC6 is highly expressed in the testes and such, and ispredicted to be relevant to normal tissue differentiation. However,HDAC6 has not been found to be related to carcinogenesis. Therefore,inhibition of HDAC6 may lead to side effects. Since the compounds of thepresent invention have extremely weak HDAC6 inhibition activity, as wellas sub-type selectivity, which TSA does not have, they are useful asnovel inhibitors. Furthermore, the tetrapeptide backbone structure ofthe compounds of the present invention can be easily changed, suggestingfurther selectivity can be conferred.

INDUSTRIAL APPLICABILITY

As described above, the compounds of the present invention show strongselective inhibitory activity towards HDAC1 and HDAC4. Accordingly, thecompounds of the present invention may be useful as pharmaceuticalagents for treating or preventing diseases associated with HDACs,particularly HDAC1 and HDAC4. The methods for producing the compounds ofthe present invention are carried out by using 2-amino-n-haloalkanoicacid as a raw material to easily synthesize various types of compounds.Consequently, use of the production methods of the present invention isexpected to contribute to the development of HDAC inhibitors withgreater selectivity.

1. A compound represented by the following formula (1):

[wherein, R₁₁, R₂₁, R₃₁, and R₄₁ independently denote hydrogen ormethyl; R₂₂, R₂₃, R₃₂, R₃₃, R₄₂, and R₄₃ independently denote ahydrogen, a linear alkyl with one to six carbon atoms, a linear alkylwith one to six carbon atoms to which a non-aromatic cyclic alkyl groupor substituted or unsubstituted aromatic ring, a hon-aromatic cyclicalkyl, or a non-aromatic cyclic alkyl group to which a non-aromaticcyclic alkyl group or a substituted or unsubstituted aromatic ring isbound; the pairs of R₂₁ and R₂₂, R₂₂ and R₂₃, R₃₁ and R₃₂, R₃₂ and R₃₃,R₄₁ and R₄₂, and R₄₂ and R₄₃ independently denote acyclic structureswithout binding or cyclic structures by binding through a linearalkylene group with a one- to five-carbon main chain, a linear alkylenegroup with a one- to five-carbon main chain comprising a branched chainwith one to six carbons, or a linear alkylene group with a one- tofive-carbon main chain comprising a ring structure of one to sixcarbons; X denotes hydrogen, a structure identical to that shown to theleft of X, a substituted or unsubstituted alkyl or aryl group in anystructure comprising a sulfur atom capable of binding with the sulfuratom in formula (1) through a disulfide bond, or a sulfur atom bindingwith the sulfur atom bonded to the terminus of R₂₂, R₂₃, R₃₂, R₃₃, R₄₂,or R₄₃, and located to the left of X, via an intramolecular disulfidebond].
 2. A histone deacetylase inhibitor that comprises the compound ofclaim 1 as an active ingredient.
 3. An apoptosis-inducing agent thatcomprises the compound of claim 1 as an active ingredient.
 4. Adifferentiation-inducing agent that comprises the compound of claim 1 asan active ingredient.
 5. An angiogenesis inhibitor that comprises thecompound of claim 1 as an active ingredient.
 6. An anti-metastatic agentcomprising the compound of claim 1 as an active ingredient.
 7. Apharmaceutical agent for treating or preventing a disease caused byhistone deacetylase 1 or 4, comprising the compound of claim 1 as anactive ingredient.
 8. The pharmaceutical agent of claim 7, wherein thedisease caused by histone deacetylase 1 or 4 is cancer, autoimmunedisease, skin disease, or infectious disease.
 9. A method for producingthe compound of claim 1, which comprises the steps of: reacting acompound represented by formula (2)

(wherein, n is same as that defined in formula (1); Hal denotes ahalogen atom selected from a chlorine atom, bromine atom, or iodineatom, or an allyl or alkylsulfoxy group useful for a free group; P₂denotes a protection group for an amino group); with a compoundrepresented by formula (3)

(wherein. R₁₁, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, R₃₃, R₄₁, R₄₂, and R₄₃ are sameas defined in formula (1); P₂ denotes a protection group for a carboxylgroup); in the presence of a peptide-bonding agent to obtain a compoundrepresented by formula (4)

(wherein n, R₁₁, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, R₃₃, R₄₁, R₄₂, R₄₃, P₁, P₂,and Hal are the same as defined above); subjecting the compoundrepresented by formula (4) to catalytic hydrogenation, acid treatment,or hydrolysis to remove P₁ and P₂; and then subjecting to cyclization inthe presence of a peptide-bonding agent to obtain a compound representedby formula (5)

(wherein n, R₁₁, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, R₃₃, R₄₁, R₄₂, R₄₃, P₁, P₂,and Hal are the same as defined above); or reacting a compoundrepresented by formula (6)

(wherein R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, R₃₃, R₄₁, R₄₂, R₄₃, and P₁ are thesame as defined above); with a compound represented by formula (7)

(wherein n, R₁₁, P₂, and Hal are the same as defined above); in thepresence of a peptide-bonding agent to obtain a compound represented byformula (8)

(wherein n, R₁₁, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, R₃₃, R₄₁, R₄₂, R₄₃, P₁, P₂,and Hal are the same as defined above); subjecting the compoundrepresented by formula (8) to catalytic hydrogenation, acid treatment,fluoride anion treatment, or hydrolysis to remove P₁ and P₂; and thensubjecting to cyclization in the presence of a peptide-bonding agent toobtain the compound represented by formula (5); following, for bothprocess, the steps of: reacting the compound represented by formula (5)with a reagent comprising sulfur atoms to obtain a compound representedby formula (9)

(wherein n, R₁₁, R₂₁, R₂₂, R₂₃, R₃₁, R₃₂, R₃₃, R₄₁, R₄₂, and R₄₃ are thesame as defined above; P₃ denotes a protection group for sulfohydrylgroup); and then treating the compound represented by formula (9) withan oxidizing agent as well as ammonia or another amine.